Maize Stress-Responsive NAC Transcription Factors and Promoter and Methods of Use

ABSTRACT

Methods and compositions for modulating plant development are provided. Polynucleotide sequences encoding ZmSNAC polypeptides are provided, as are the amino acid sequences of the encoded polypeptides. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and/or shoot development, modulating senescence, modulating seed size and/or weight, and modulating tolerance of plants to abiotic stress. Transformed plants, plant cells, tissues, and seed are also provided. A stress-inducible ZmSNAC1 promoter is also provided.

CROSS REFERENCE

This application is a continuation of Utility patent application Ser.No. 12/254,268 filed Oct. 20, 2008 which claims priority to ProvisionalPatent Application Ser. No. 60/981,228 filed Oct. 19, 2007, both ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of genetic manipulation of plants,particularly the modulation of gene activity to affect plant developmentand growth.

BACKGROUND OF THE INVENTION

Drought tolerance is a complex trait, controlled by multiple genes. Geneswitches such as transcription factors, which can have significant andspecific effects on plant physiology, are desirable modulation targets.Stress-responsive NAC transcription factors (SNACs) may controlexpression of numerous downstream genes important for adaptation todrought stresses and may ultimately enhance drought tolerance throughone or more mechanisms such as stomatal aperture reduction, delayedsenescence, and increased sink and source strength. It has been shownthat ABA- and drought-responsive NACs can enhance drought tolerance inArabidopsis (Tran, et al., (2004) Plant Cell 16:2481-2498) and rice (Hu,et al., (2006) PNAS 103(35):12987-12992). Therefore, SNAC genes frommaize may also be used individually or in combination with other genesto enhance drought tolerance.

Expression of heterologous DNA sequences in a plant host is dependentupon the presence of operably linked regulatory elements that arefunctional within the plant host. Choice of the regulatory element willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where continuous expression is desired throughoutthe cells of a plant and/or throughout development, constitutivepromoters are utilized. In contrast, where gene expression in responseto a stimulus is desired, inducible promoters are the regulatory elementof choice. Where expression in specific tissues or organs is desired,tissue-specific or tissue-preferred promoters may be used. That is, theymay drive expression exclusively or preferentially in specific tissuesor organs. Such tissue-specific promoters may be temporally constitutiveor inducible. In any case, additional regulatory sequences upstreamand/or downstream from a core promoter sequence may be included inexpression constructs of transformation vectors to bring about varyinglevels of expression of heterologous nucleotide sequences in atransgenic plant.

As this field develops and more genes become accessible, a greater needexists for plants transformed with multiple genes. These multipleexogenous genes typically need to be controlled by separate regulatorysequences. Further, some genes should be regulated constitutivelywhereas other genes should be expressed at certain developmental stagesor locations in the transgenic organism. Accordingly, a variety ofregulatory sequences having diverse effects is needed.

Multiple regulatory sequences are also needed in order to avoidundesirable molecular interactions which can result from using the sameregulatory sequence to control more than one gene.

The inventor herein discloses the isolation and characterization of apromoter associated with a transcription factor which can serve as aregulatory element for expression of isolated nucleotide sequences ofinterest, thereby impacting various traits in plants. Alternatively oradditionally, the promoter may be used to drive scorable markers.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods of the invention comprise and employ maize SNACtranscription factors that are involved in modulating plant development,morphology and physiology.

Compositions further include expression cassettes and vectors comprisingthe ZmSNAC sequences of the invention and plants, plant cells and plantparts in which ZmSNAC expression is modified. The plants, plant cellsand plant parts of the invention may exhibit enhanced abiotic stresstolerance through such phenotypic changes as delayed leaf rolling,reduced transpiration rate, increased stomatal closure and improved cellmembrane stability, all relative to a plant, plant cell or plant partnot modified per the invention.

Methods are provided for modifying the level of a ZmSNAC polypeptide ina plant comprising introducing into the plant a selected polynucleotide.The polynucleotide may be introduced within a construct designed tomodulate the level or activity of the ZmSNAC polypeptide throughout theplant or in targeted tissues or at targeted developmental stages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an amino acid alignment of NAC transcription factorsfrom various species. Included are ANAC (At1g52890, SEQ ID NO: 12);ATAF2 (At5g08790, SEQ ID NO: 13); ATAF1 (At1g01720, SEQ ID NO: 14); NAP(At1g69490, SEQ ID NO: 15); AtNAM (At1g52880, SEQ ID NO: 16); TIP(At5g24590, SEQ ID NO: 17); CUC2 (At5g53950, SEQ ID NO: 18); CUC1(At3g15170, SEQ ID NO: 19); CUC3 (At1g76420, SEQ ID NO: 20); ArabidopsisNAC1 (At1g56010, SEQ ID NO: 21); ZmSNAC1 (SEQ ID NO: 2); ZmSNAC2 (SEQ IDNO: 4); ZmSNAC3 (SEQ ID NO: 6); ZmSNAC5 (SEQ ID NO: 10); ZmSNAC4 (SEQ IDNO: 8) and a consensus (SEQ ID NO: 22).

FIG. 2 shows GUS expression driven by the ZmSNAC1 promoter.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 and 2 provide nucleotide and amino acid sequences forZmSNAC1.

SEQ ID NO: 3 and 4 provide nucleotide and amino acid sequences forZmSNAC2.

SEQ ID NO: 5 and 6 provide nucleotide and amino acid sequences forZmSNAC3.

SEQ ID NO: 7 and 8 provide nucleotide and amino acid sequences forZmSNAC4.

SEQ ID NO: 9 and 10 provide nucleotide and amino acid sequences forZmSNAC5.

SEQ ID NO: 11 provides ZmSNAC1 promoter sequence.

SEQ ID NOS: 12 through 21 provide Arabidopsis NAC proteins as noted inthe brief description of the drawings.

SEQ ID NO: 22 provides a consensus of Arabidopsis and Zea mays NACproteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully with reference tothe accompanying drawings, in which some, but not all, embodiments ofthe invention are shown. Indeed, the invention may be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will satisfy applicable legal requirements. Likenumbers refer to like elements throughout.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Compositions

Compositions of the invention include maize SNAC transcription factorpolypeptides and polynucleotides that are involved in modulating plantdevelopment, morphology and physiology. In particular, the presentinvention provides for isolated polynucleotides comprising nucleotidesequences encoding the amino acid sequences shown in SEQ ID NO: 2, 4, 6,8 and 10. Further provided are isolated polypeptides having an aminoacid sequence encoded by a polynucleotide described herein, for examplethose set forth in SEQ ID NO: 1, 3, 5, 7 and 9.

NAC transcription factors are encoded by genes present in a wide rangeof plant species, the name being derived from the NAM (no apicalmeristem; Souer, et al., (1996) Cell 85:159-170), ATAF1,2 and CUC2(cup-shaped cotyledon 2; Aida, et al., (1997) Plant Cell 9:841-857)transcription factors. Expression patterns of NAC transcription factors,and the mutant phenotypes conferred by modulation of their expression,are similar. A highly conserved N-terminal DNA-binding domain has beenidentified and its structure has been characterized (Ernst, et al.,(2004) EMBO Reports 5(3):297-303). The more diverse C-terminal regionscomprise transcriptional activation domains (Xie, et al., (2000) GenesDev. 14:3024-3036; Duval, et al., (2002) Plant Mol. Bio. 50:237-248).NAC transcription factors have been shown to interact with numerousgenes involved in meristem formation, organ differentiation, auxinsignalling, root growth and in biotic and abiotic stress response (see,review by Olsen, et al., (2005) Trends in Plant Science 10(2)1360-1385).A NAC recognition sequence (NACRS), and a core binding sequence, havebeen identified in Arabidopsis (Tran, et al., (2004) Plant Cell16:2481-2498).

The invention encompasses isolated or substantially purifiedpolynucleotide or protein compositions. An “isolated” or “purified”polynucleotide or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the polynucleotide or protein as found in itsnaturally occurring environment. Thus, an isolated or purifiedpolynucleotide or protein is substantially free of other cellularmaterial or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Optimally, an “isolated” polynucleotide is freeof sequences (optimally protein encoding sequences) that naturally flankthe polynucleotide (i.e., sequences located at the 5′ and 3′ ends of thepolynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A protein that is substantially free ofcellular material includes preparations of protein having less thanabout 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein.When the protein of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteinsencoded thereby are also encompassed by the present invention. By“fragment” is intended a portion of the polynucleotide or a portion ofthe amino acid sequence and hence protein encoded thereby. Fragments ofa polynucleotide may encode protein fragments that retain the biologicalactivity of the native protein. Alternatively, fragments of apolynucleotide that are useful as hybridization probes generally do notencode fragment proteins retaining biological activity. Thus, fragmentsof a nucleotide sequence may range from at least about 20 nucleotides,about 50 nucleotides, about 100 nucleotides and up to the full-lengthpolynucleotide encoding the proteins of the invention.

A fragment of a ZmSNAC polynucleotide that encodes a biologically activeportion of a NAC transcription factor of the invention will encode atleast 15, 25, 30, 50, 100, 150, 200, 225, 250, 275, 300 or 339contiguous amino acids, or up to the total number of amino acids presentin a full-length polypeptide of the invention. Fragments of a SNACpolynucleotide that are useful as hybridization probes or PCR primersgenerally need not encode a biologically active polypeptide.

Thus, a fragment of a ZmSNAC polynucleotide may encode a biologicallyactive portion of a SNAC transcription factor, or it may be a fragmentthat can be used as a hybridization probe or PCR primer using methodsdisclosed below. A biologically active portion of a SNAC transcriptionfactor can be prepared by isolating a portion of one of the ZmSNACpolynucleotides of the invention, expressing the encoded portion of theZmSNAC protein (e.g., by recombinant expression in vitro), and assessingthe activity of the encoded portion. Activity could be assessed by DNAbinding activity or protein interaction studies, and/or by expressingthe ZMSNAC or portions of the protein in transgenic plants andevaluating the plants for a phenotype.

Polynucleotides that are fragments of a ZmSNAC nucleotide sequencecomprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 800, 900 or 1000 contiguous nucleotides,or up to the number of nucleotides present in a full-length ZmSNACpolynucleotide disclosed herein.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively. For polynucleotides,conservative variants include those which, because of the degeneracy ofthe genetic code, do not change the encoded amino acid sequence.Naturally occurring variants such as these can be identified with theuse of well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a ZmSNAC protein of theinvention. Generally, variants of a particular polynucleotide of theinvention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to that particular polynucleotide as determined bysequence alignment programs and parameters described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, isolated polynucleotides that encodea polypeptide with a given percent sequence identity to the polypeptideof SEQ ID NO: 2, 4, 6, 8 or 10 are disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the nativeprotein by deletion or addition of one or more amino acids at one ormore sites in the native protein and/or substitution of one or moreamino acids at one or more sites in the native protein. Certain variantproteins encompassed by the present invention are biologically active,that is, they continue to possess the desired biological activity of thenative protein, i.e., transcription factor activity, as describedherein. Such variants may result from, for example, genetic polymorphismor human manipulation. Biologically active variants of a native SNACprotein of the invention will have at least about 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the amino acid sequence for thenative protein as determined by sequence alignment programs andparameters described elsewhere herein. A biologically active variant ofa protein of the invention may differ from that protein by as few as1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, asfew as 4, 3, 2 or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations and insertions. Methodsfor such manipulations are generally known in the art. For example,amino acid sequence variants and fragments of the SNAC proteins can beprepared by mutations in the DNA. Methods for mutagenesis andpolynucleotide alterations are well known in the art. See, for example,Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel, et al.,(1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walkerand Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York) and the references cited therein. Guidanceas to appropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model ofDayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be optimal.

Thus, the genes and polynucleotides of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass naturally occurring proteins as wellas variations and modified forms thereof. Such variants will continue topossess the desired transcription factor activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure.

The deletions, insertions and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by assaying for DNA binding activity or protein-proteininteractions, the activation of gene expression in transient studies oran effect on gene expression in transgenic plants.

Variant polynucleotides and proteins also encompass sequences andproteins derived from a mutagenic or recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different NAC codingsequences can be manipulated to create a new NAC polypeptide possessingthe desired properties. In this manner, libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest may be shuffled between a ZmSNAC gene of theinvention and other known NAC genes to obtain a new gene coding for aprotein with an improved property of interest. Strategies for such DNAshuffling are known in the art. See, for example, Stemmer (1994) Proc.Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391;Crameri, et al., (1997) Nature Biotech. 15:436-438; Moore, et al.,(1997) J. Mol. Biol. 272:336-347; Zhang, et al., (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri, et al., (1998) Nature 391:288-291;PCT Publication Number WO97/20078 and U.S. Pat. Nos. 5,605,793 and5,837,458.

By “promoter” is intended a regulatory region of DNA usually comprisinga TATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular polynucleotide sequence. A promoter may additionally compriseother recognition sequences generally positioned upstream or 5′ to theTATA box, referred to as upstream promoter elements, which influence thetranscription initiation rate. The promoter sequences of the presentinvention regulate (i.e., repress or activate) transcription.

The polynucleotides of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,more particularly other monocots. In this manner, methods such as PCR,hybridization and the like can be used to identify such sequences basedon their sequence homology to the sequences set forth herein. Sequencesisolated based on their sequence identity to the entire NAC sequencesset forth herein or to variants and fragments thereof are encompassed bythe present invention. Such sequences include sequences that areorthologs of the disclosed sequences. “Orthologs” is intended to meangenes derived from a common ancestral gene and which are found indifferent species as a result of speciation. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequenceidentity. Functions of orthologs are often highly conserved amongspecies. Thus, isolated polynucleotides that encode a NAC protein andwhich hybridize under stringent conditions to the SNAC sequencesdisclosed herein, or to variants or fragments or complements thereof,are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any plant of interest. Methods for designingPCR primers and PCR cloning are generally known in the art and aredisclosed in Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methodsand Applications (Academic Press, New York); Innis and Gelfand, eds.(1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand,eds. (1999) PCR Methods Manual (Academic Press, New York). Known methodsof PCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers and the like.

In hybridization techniques, all or part of a known polynucleotide isused as a probe that selectively hybridizes to other correspondingpolynucleotides present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosenorganism. The hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as ³²P or any other detectable marker.Thus, for example, probes for hybridization can be made by labelingsynthetic oligonucleotides based on the ZmSNAC polynucleotides of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

For example, an entire ZmSNAC polynucleotide disclosed herein, or one ormore portions thereof, may be used as a probe capable of specificallyhybridizing to corresponding NAC polynucleotides. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among NAC polynucleotide sequences and areoptimally at least about 10 nucleotides in length and most optimally atleast about 20 nucleotides in length. Such probes may be used to amplifycorresponding NAC polynucleotides from a chosen plant by PCR. Thistechnique may be used to isolate additional coding sequences from adesired plant or as a diagnostic assay to determine the presence ofcoding sequences in a plant. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook, et al., (1989) Molecular Cloning:A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C. and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.The duration of the wash time will be at least a length of timesufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is optimal to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York) and Ausubel, et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See, Sambrook, et al., (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity” and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twopolynucleotides. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, (1988) CABIOS 4:11-17; the local alignmentalgorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482; the globalalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-453; the search-for-local alignment method of Pearson and Lipman,(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul, (1990) Proc. Natl. Acad. Sci. USA 872264, modified as inKarlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins, et al.,(1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153;Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al.,(1992) CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-331. The ALIGN program is based on the algorithm of Myers andMiller, (1988) supra. A PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4 can be used with the ALIGN programwhen comparing amino acid sequences. The BLAST programs of Altschul, etal., (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlinand Altschul, (1990) supra. BLAST nucleotide searches can be performedwith the BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul, et al., (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See, Altschul, et al., (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See, www.ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2 and theBLOSUM62 scoring matrix.

GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the GCG Wisconsin Genetics Software Packageis BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci.USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo polynucleotides or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The invention further provides plants having altered levels and/oractivities of the ZmNAC polypeptides of the invention. In someembodiments, a construct directing increased expression of the sequencesof the invention is stably incorporated into the genome of a plant ofthe invention. Other embodiments provide plants that are geneticallymodified at a native genomic locus encoding a SNAC polypeptide of theinvention. By “native genomic locus” is intended a naturally occurringgenomic sequence. The genomic locus may be modified to increase, reduceor eliminate the activity of the SNAC polypeptide. The term “geneticallymodified” as used herein refers to a plant or plant part that ismodified in its genetic information by the introduction of one or moreforeign polynucleotides, and the introduction of the foreignpolynucleotide leads to a phenotypic change in the plant. By “phenotypicchange” is intended a measurable change in one or more cell, tissue ororgan functions. For example, plants having a genetic modification atthe genomic locus encoding a ZmSNAC polypeptide can show reduced oreliminated expression or activity of the NAC polypeptide. Variousmethods to generate such a genetically modified genomic locus aredescribed elsewhere herein, as are the variety of phenotypes that canresult from the modulation of the level and/or activity of one or moreof the ZmSNAC sequences of the invention.

As used herein, the term plant includes reference to whole plants, plantparts or organs (e.g., leaves, stems, roots), plant cells and seeds andprogeny of same. Plant cell, as used herein, includes, withoutlimitation, cells obtained from or found in seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores, as well as plantprotoplasts and plant cell tissue cultures, plant calli, plant clumpsand plant cells that are intact in plants or parts of plants such asembryos, pollen, ovules, seeds, leaves, flowers, branches, fruit,kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain andthe like. As used herein, “grain” refers to the mature seed produced bycommercial growers for purposes other than growing or reproducing thespecies. Progeny, variants and mutants of the regenerated plants arealso included within the scope of the invention, provided that theseparts comprise the introduced nucleic acid sequences.

Methods

I. Providing Sequences

The sequences of the present invention can be introduced into andexpressed in a host cell such as bacteria, yeast, insect, mammalian oroptimally plant cells. It is expected that those of skill in the art areknowledgeable in the numerous systems available for the introduction ofa polypeptide or a nucleotide sequence of the present invention into ahost cell. No attempt to describe in detail the various methods knownfor providing proteins in prokaryotes or eukaryotes will be made.

By “host cell” is meant a cell which comprises a heterologous nucleicacid sequence of the invention. Host cells may be prokaryotic cells suchas E. coli or eukaryotic cells such as yeast, insect, amphibian ormammalian cells. Host cells can also be monocotyledonous ordicotyledonous plant cells. In certain embodiments, the monocotyledonoushost cell is a maize host cell.

The use of the term “polynucleotide” is not intended to limit thepresent invention to polynucleotides comprising DNA. Those of ordinaryskill in the art will recognize that polynucleotides can compriseribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures and the like.

The ZmSNAC polynucleotides of the invention can be provided inexpression cassettes for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa ZmSNAC polynucleotide of the invention. “Operably linked” is intendedto mean a functional linkage between two or more elements. For example,an operable linkage between a polynucleotide of interest and aregulatory sequence (i.e., a promoter) is a functional link that allowsfor expression of the polynucleotide of interest. Operably linkedelements may be contiguous or non-contiguous. When used to refer to thejoining of two protein coding regions, “operably linked” means that thecoding regions are in the same reading frame. The cassette may containat least one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes. An expression cassette may be provided with aplurality of restriction sites and/or recombination sites for insertionof the ZmSNAC polynucleotide to be under the transcriptional regulationof the regulatory regions. The expression cassette may additionallycontain selectable marker genes.

In certain embodiments, the expression cassette will include, in the5′-3′ direction of transcription, a transcriptional and translationalinitiation region, a ZmSNAC polynucleotide of the invention, and atranscriptional and translational termination region functional inplants. The regulatory regions (i.e., promoters, transcriptionalregulatory regions, and translational termination regions) and/or theZmSNAC polynucleotide of the invention may be native/analogous to thehost cell or to each other. Alternatively, the regulatory regions and/orthe ZmNAC polynucleotide of the invention may be heterologous to thehost cell or to each other. As used herein with reference to a sequence,“heterologous” means a sequence that originates from a foreign species,or, if from the same species, is substantially modified from its nativeform in composition and/or genomic locus by deliberate humanintervention. For example, a promoter operably linked to a heterologouspolynucleotide is from a species different from the species from whichthe polynucleotide was derived, or, if from the same/analogous species,one or both are substantially modified from their original form and/orgenomic locus or the promoter is not the native promoter for theoperably-linked polynucleotide. As used herein, a chimeric genecomprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

While heterologous promoters can be used to express the ZmSNACsequences, the native promoter sequences or other NAC promoters may alsobe used. Such constructs can change expression levels of ZmSNACsequences in the plant or plant cell. Thus, the phenotype of the plantor plant cell can be altered.

The termination region may be native with the transcriptional initiationregion, may be native with the operably-linked ZmSNAC polynucleotide ofinterest, may be native with the plant host, or may be derived fromanother source, i.e., foreign or heterologous with reference to thepromoter, the ZmSNAC polynucleotide of interest, the plant host, or anycombination thereof. Convenient termination regions are available fromthe Ti-plasmid of A. tumefaciens, such as the octopine synthase andnopaline synthase termination regions. See also, Guerineau, et al.,(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674;Sanfacon, et al., (1991) Genes Dev. 5:141-149; Mogen, et al., (1990)Plant Cell 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas,et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987)Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831 and 5,436,391 and Murray, et al., (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats and other such well-characterized sequences thatmay be deleterious to gene expression. The G-C content of the sequencemay be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak, et al., (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling, et al., (1987) Nature 325:622-625); tobacco mosaic virusleader (TMV) (Gallie, et al., (1989) in Molecular Biology of RNA, ed.Cech (Liss, New York), pp. 237-256) and maize chlorotic mottle virusleader (MCMV) (Lommel, et al., (1991) Virology 81:382-385). See also,Della-Cioppa, et al., (1987) Plant Physiol. 84:965-968. Other methodsknown to enhance translation can also be utilized.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su, et al., (2004)Biotechnol Bioeng 85:610-9 and Fetter, et al., (2004) Plant Cell16:215-28), cyan fluorescent protein (CYP) (Bolte, et al., (2004) J.Cell Science 117:943-54 and Kato, et al., (2002) Plant Physiol129:913-42) and yellow fluorescent protein (PhiYFP™ from Evrogen, see,Bolte, et al., (2004) J. Cell Science 117:943-54). For additionalselectable markers, see generally, Yarranton, (1992) Curr. Opin.Biotech. 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad.Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; Reznikoff,(1992) Mol. Microbiol. 6:2419-2422; Barkley, et al., (1980) in TheOperon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566; Brown, et al.,(1987) Cell 49:603-612; Figge, et al., (1988) Cell 52:713-722; Deuschle,et al., (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst, et al.,(1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle, et al., (1990)Science 248:480-483; Gossen (1993) Ph.D. Thesis, University ofHeidelberg; Reines, et al., (1993) Proc. Natl. Acad. Sci. USA90:1917-1921; Labow, et al., (1990) Mol. Cell. Biol. 10:3343-3356;Zambretti, et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim,et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski, etal., (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, (1989)Topics Mol. Struc. Biol. 10:143-162; Degenkolb, et al., (1991)Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt, et al., (1988)Biochemistry 27:1094-1104; Bonin, (1993) Ph.D. Thesis, University ofHeidelberg; Gossen, et al., (1992) Proc. Natl. Acad. Sci. USA89:5547-5551; Oliva, et al., (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka, et al., (1985) Handbook of ExperimentalPharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill, et al., (1988)Nature 334:721-724. Such disclosures are herein incorporated byreference. The above list of selectable marker genes is not meant to belimiting; any selectable marker gene can be used in the presentinvention.

Certain other marker genes for plant transformation require screening ofpresumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These marker genes are particularly useful to quantifyor visualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, (1987)Plant Mol. Biol. Rep. 5.287 and U.S. Pat. No. 5,599,670; Teeri, et al.,(1989) EMBO J. 8:343, Koncz, et al., (1987) Proc. Natl. Acad. Sci.U.S.A. 84:131, De Block, et al., (1984) EMBO J. 3:1681. Another approachto the identification of relatively rare transformation events has beenuse of a gene that encodes a dominant constitutive regulator of the Zeamays anthocyanin pigmentation pathway. Ludwig, et al., (1990) Science247: 449.

A number of promoters can be used in the practice of the invention,including the native promoter of a polynucleotide sequence of interest.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, inducible, tissue-preferred orother promoters for expression in plants.

Such constitutive promoters include, for example, the core promoter ofthe Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell,et al., (1985) Nature 313:810-812); rice actin (McElroy, et al., (1990)Plant Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Mol.Biol. 12:619-632 and Christensen, et al., (1992) Plant Mol. Biol.18:675-689); pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-588);MAS (Velten, et al., (1984) EMBO J. 3:2723-2730); ALS promoter (U.S.Pat. No. 5,659,026), dMMV (double-enhanced version of the mirabilismosaic virus promoter; see, Dey and Maiti, (1999) Plant MolecularBiology 40(5):771-782), LESVBV (enhanced strawberry vein banding viruspromoter; see, US Patent Application Publication Number 2002/0182593)and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced ZmNACexpression within a particular plant tissue. Tissue-preferred promotersinclude Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kawamata, etal., (1997) Plant Cell Physiol. 38(7):792-803; Hansen, et al., (1997)Mol. Gen Genet. 254(3):337-343; Russell, et al., (1997) Transgenic Res.6(2):157-168; Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341;Van Camp, et al., (1996) Plant Physiol. 112(2):525-535; Canevascini, etal., (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994)Plant Cell Physiol. 35(5):773-778; Lam, (1994) Results Probl. CellDiffer. 20:181-196; Orozco, et al., (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590 and Guevara-Garcia, et al., (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression. See, also, US Patent Application Number 2003/0074698, hereinincorporated by reference.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138; Baszczynski, et al., (1988)Nucl. Acid Res. 16:4732; Mitra, et al., (1994) Plant Molecular Biology26:35-93; Kayaya, et al., (1995) Molecular and General Genetics248:668-674 and Matsuoka, et al., (1993) Proc. Natl. Acad. Sci. USA90(20):9586-9590. Senecence regulated promoters are also of use, suchas, SAM22 (Crowell, et al., (1992) Plant Mol. Biol. 18:459-466). See,also, U.S. Pat. No. 5,689,042, herein incorporated by reference.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire, et al., (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger, et al.,(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens) and Miao, etal., (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also, Bogusz, et al., (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus and in both instances root-specific promoter activitywas preserved. Leach and Aoyagi, (1991) describe their analysis of thepromoters of the highly expressed roIC and roID root-inducing genes ofAgrobacterium rhizogenes (see, Plant Science (Limerick) 79(1):69-76).They concluded that enhancer and tissue-preferred DNA determinants aredissociated in those promoters. Teeri, et al., (1989) used gene fusionto lacZ to show that the Agrobacterium T-DNA gene encoding octopinesynthase is especially active in the epidermis of the root tip and thatthe TR2′ gene is root specific in the intact plant and stimulated bywounding in leaf tissue, an especially desirable combination ofcharacteristics for use with an insecticidal or larvicidal gene (see,EMBO J. 8(2):343-350). The TR1′ gene, fused to nptII (neomycinphosphotransferase II) showed similar characteristics. Additionalroot-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster,et al., (1995) Plant Mol. Biol. 29(4):759-772); roIB promoter (Capana,et al., (1994) Plant Mol. Biol. 25(4):681-691; and the CRWAQ81root-preferred promoter with the ADH first intron (US Patent ApplicationPublication Number 2005/0097633). See also, U.S. Pat. Nos. 5,837,876;5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

“Seed-preferred” promoters refers to those promoters active during seeddevelopment and may include expression in seed initials or relatedmaternal tissue. Such seed-preferred promoters include, but are notlimited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDazein); milps (myo-inositol-1-phosphate synthase) (see, WO 00/11177 andU.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zeinis an endosperm-specific promoter. Globulin-1 (Glob-1) is arepresentative embryo-specific promoter. For dicots, seed-specificpromoters include, but are not limited to, bean β-phaseolin, napin,β-conglycinin, soybean lectin, cruciferin and the like. For monocots,seed-specific promoters include, but are not limited to, maize 15 kDazein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1 andshrunken 2. See also, WO 00/12733, where seed-preferred promoters fromend1 and end2 genes are disclosed, herein incorporated by reference.Additional embryo specific promoters are disclosed in Sato, et al.,(1996) Proc. Natl. Acad. Sci. 93:8117-8122; Nakase, et al., (1997) PlantJ 12:235-46; and Postma-Haarsma, et al., (1999) Plant Mol. Biol.39:257-71. Additional endosperm specific promoters are disclosed inAlbani, et al., (1984) EMBO 3:1405-15; Albani, et al., (1999) Theor.Appl. Gen. 98:1253-62; Albani, et al., (1993) Plant J. 4:343-55; Mena,et al., (1998) The Plant Journal 116:53-62 and Wu, et al., (1998) PlantCell Physiology 39:885-889.

Also of interest are promoters active in meristem regions, such asdeveloping inflorescence tissues and promoters which drive expression ator about the time of anthesis or early kernel development. This mayinclude, for example, the maize Zag promoters, including Zag1 and Zag2(see, Schmidt, et al., (1993) The Plant Cell 5:729-37; GenBank X80206;Theissen, et al., (1995) Gene 156:155-166 and U.S. patent applicationSer. No. 10/817,483); maize Zap promoter (also known as ZmMADS; U.S.patent application Ser. No. 10/387,937; WO 03/078590); maize ckx1-2promoter (US Patent Application Publication Number 2002/0152500 A1; WO02/0078438); maize eep1 promoter (U.S. patent application Ser. No.10/817,483); maize end2 promoter (U.S. Pat. No. 6,528,704 and U.S.patent application Ser. No. 10/310,191); maize lec1 promoter (U.S.patent application Ser. No. 09/718,754); maize F3.7 promoter(Baszczynski, et al., (1997) Maydica 42:189-201); maize tb1 promoter(Hubbarda, et al., (2002) Genetics 162:1927-1935 and Wang, et al.,(1999) Nature 398:236-239); maize eep2 promoter (U.S. patent applicationSer. No. 10/817,483); maize thioredoxinH promoter (U.S. ProvisionalPatent Application No. 60/514,123); maize Zm40 promoter (U.S. Pat. No.6,403,862 and WO 01/2178); maize mLIP15 promoter (U.S. Pat. No.6,479,734); maize ESR promoter (U.S. patent application Ser. No.10/786,679); maize PCNA2 promoter (U.S. patent application Ser. No.10/388,359); maize cytokinin oxidase promoters (U.S. patent applicationSer. No. 11/094,917); promoters disclosed in Weigal, et al., (1992) Cell69:843-859; Accession Number AJ131822; Accession Number Z71981;Accession Number AF049870 and shoot-preferred promoters disclosed inMcAvoy, et al., (2003) Acta Hort. (ISHS) 625:379-385. Other dividingcell or meristematic tissue-preferred promoters that may be of interesthave been disclosed in Ito, et al., (1994) Plant Mol. Biol. 24:863-878;Regad, et al., (1995) Mo. Gen. Genet. 248:703-711; Shaul, et al., (1996)Proc. Natl. Acad. Sci. 93:4868-4872; Ito, et al., (1997) Plant J.11:983-992 and Trehin, et al., (1997) Plant Mol. Biol. 35:667-672, allof which are hereby incorporated by reference herein.

Inflorescence-preferred promoters include the promoter of chalconesynthase (Van der Meer, et al., (1990) Plant Mol. Biol. 15:95-109),LAT52 (Twell, et al., (1989) Mol. Gen. Genet. 217:240-245), pollenspecific genes (Albani, et al., (1990) Plant Mol Biol. 15:605, Zm13(Buerrero, et al., (1993) Mol. Gen. Genet. 224:161-168), maizepollen-specific gene (Hamilton, et al., (1992) Plant Mol. Biol.18:211-218), sunflower pollen expressed gene (Baltz, et al., (1992) ThePlant Journal 2:713-721) and B. napus pollen specific genes (Arnoldo, etal., (1992) J. Cell. Biochem, Abstract Number Y101204).

Stress-inducible promoters include salt-inducible orwater-stress-inducible promoters such as P5CS (Zang, et al., (1997)Plant Sciences 129:81-89); cold-inducible promoters, such as, cor15a(Hajela, et al., (1990) Plant Physiol. 93:1246-1252), cor15b (Wlihelm,et al., (1993) Plant Mol Biol 23:1073-1077), wsc120 (Ouellet, et al.,(1998) FEBS Lett. 423-324-328), ci7 (Kirch, et al., (1997) Plant MolBiol. 33:897-909), ci21A (Schneider, et al., (1997) Plant Physiol.113:335-45); drought-inducible promoters, such as, Trg-31 (Chaudhary, etal., (1996) Plant Mol. Biol. 30:1247-57); osmotic inducible promoters,such as, Rab17 (Vilardell, et al., (1991) Plant Mol. Biol. 17:985-93)and osmotin (Raghothama, et al., (1993) Plant Mol Biol 23:1117-28) andheat inducible promoters, such as, heat shock proteins (Barros, et al.,(1992) Plant Mol. 19:665-75; Marrs, et al., (1993) Dev. Genet. 14:27-41)and smHSP (Waters, et al., (1996) J. Experimental Botany 47:325-338).Other stress-inducible promoters include rip2 (U.S. Pat. No. 5,332,808and US Patent Application Publication Number 2003/0217393), rab17 (Busk,(1997) Plant J 11(6):1285-1295) and rd29a (Yamaguchi-Shinozaki, et al.,(1993) Mol. Gen. Genetics 236:331-340). Manipulation of ZmNAC expressionto improve abiotic stress tolerance may involve the use oftissue-preferred and/or stress-responsive promoters.

Stress-insensitive promoters can also be used in the methods of theinvention. This class of promoters, as well as representative examples,are further described elsewhere herein.

Nitrogen-responsive promoters can also be used in the methods of theinvention. Such promoters include, but are not limited to, the 22 kDaZein promoter (Spena, et al., (1982) EMBO J 1:1589-1594 and Muller, etal., (1995) J. Plant Physiol 145:606-613); the 19 kDa zein promoter(Pedersen, et al., (1982) Cell 29:1019-1025); the 14 kDa zein promoter(Pedersen, et al., (1986) J. Biol. Chem. 261:6279-6284), the b-32promoter (Lohmer, et al., (1991) EMBO J 10:617-624) and the nitritereductase (NiR) promoter (Rastogi, et al., (1997) Plant Mol Biol.34(3):465-76 and Sander, et al., (1995) Plant Mol Biol. 27(1):165-77).For a review of consensus sequences found in nitrogen-induced promoters,see for example, Muller, et al., (1997) The Plant Journal 12:281-291.

Chemically-regulated promoters can be used to modulate the expression ofa gene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemically-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemically-induciblepromoters are known in the art and include, but are not limited to, themaize In2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides and thetobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Additional inducible promoters include heat shock promoters, such asGmhsp17.5-E (soybean) (Czarnecka, et al., (1989) Mol Cell Biol.9(8):3457-3463); APX1 gene promoter (Arabidopsis) (Storozhenko, et al.,(1998) Plant Physiol. 118(3):1005-1014): Ha hsp17.7 G4 (Helianthusannuus) (Almoguera, et al., (2002) Plant Physiol. 129(1):333-341 andMaize Hsp70 (Rochester, et al., (1986) EMBO J. 5: 451-8.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotides or polypeptides intoplants are known in the art and include, but are not limited to, stabletransformation methods, transient transformation methods andvirus-mediated methods.

“Stable transformation” is intended to mean that the introducednucleotide construct integrates into the genome of the plant and iscapable of being inherited by the progeny thereof. “Transienttransformation” is intended to mean that a sequence is introduced intothe plant but is only temporarily expressed or present in the plant.

Transformation protocols, as well as protocols for introducingpolypeptides or polynucleotide sequences into plants, may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway, etal., (1986) Biotechniques 4:320-334), electroporation (Riggs, et al.,(1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski, et al., (1984) EMBO J. 3:2717-2722)and ballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244 and5,932,782; Tomes, et al., (1995) in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); McCabe, et al., (1988) Biotechnology 6:923-926) and Lec1transformation (WO 00/28058). Also see, Weissinger, et al., (1988) Ann.Rev. Genet. 22:421-477; Sanford, et al., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.87:671-674 (soybean); McCabe, et al., (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen, (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh, et al., (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta, et al., (1990) Biotechnology 8:736-740(rice); Hoque, et al., (2005) Plant Cell Tissue & Organ Culture82(1):45-55 (rice); Sreekala, et al., (2005) Plant Cell Reports24(2):86-94 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, New York), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens), all of which are herein incorporated byreference.

In specific embodiments, the ZmSNAC sequences of the invention can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the ZmSNAC protein or variants and fragments thereofdirectly into the plant or the introduction of a ZmSNAC transcript intothe plant. Such methods include, for example, microinjection or particlebombardment. See, for example, Crossway, et al., (1986) Mol Gen. Genet.202:179-185; Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, et al.,(1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, et al., (1994) TheJournal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the ZmSNAC polynucleotide canbe transiently transformed into the plant using other techniques knownin the art. Such techniques include viral vector system and theprecipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethyleneimine (PEI;Sigma #P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that a ZmSNAC polynucleotide of the inventionmay be initially synthesized as part of a viral polyprotein, which latermay be processed by proteolysis in vivo or in vitro to produce thedesired recombinant protein. Further, it is recognized that promotersuseful for the invention also encompass promoters utilized fortranscription by viral RNA polymerases. Methods for introducingpolynucleotides into plants and expressing a protein encoded therein,involving viral DNA or RNA molecules, are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367,5,316,931 and Porta, et al., (1996) Molecular Biotechnology 5:209-221,herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855 and WO99/25853 and U.S. Pat. Nos. 6,187,994; 6,552,248; 6,624,297; 6,331,661;6,262,341; 6,541,231; 6,664,108; 6,300,545; 6,528,700 and 6,911,575, allof which are herein incorporated by reference. Briefly, thepolynucleotide of the invention can be contained in a transfer cassetteflanked by two non-recombinogenic recombination sites. The transfercassette is introduced into a plant having stably incorporated into itsgenome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional means. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be pollinatedwith either the same transformed strain or different strains and theresulting progeny having expression of the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that expression of the desired phenotypic characteristic isstably maintained and inherited and then seeds harvested to ensure thatexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into its genome.

Pedigree breeding starts with the crossing of two genotypes, such as anelite line of interest and one other inbred line having one or moredesirable characteristics (i.e., having stably incorporated apolynucleotide of the invention, having a modulated activity and/orlevel of the polypeptide of the invention, etc) which complements theelite line of interest. If the two original parents do not provide allthe desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations the heterozygous condition gives way to homogeneous lines asa result of self-pollination and selection. Typically in the pedigreemethod of breeding, five or more successive filial generations ofselfing and selection are practiced: F1→F2; F2→F3; F3→F4; F4→F₅, etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed inbred. In specificembodiments, the inbred line comprises homozygous alleles at about 95%or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify anelite line of interest and a hybrid that is made using the modifiedelite line. Backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, an F1, such as acommercial hybrid, is created. This commercial hybrid may be backcrossedto one of its parent lines to create a BC1 or BC2. Progeny are selfedand selected so that the newly developed inbred has many of theattributes of the recurrent parent and yet several of the desiredattributes of the non-recurrent parent. This approach leverages thevalue and strengths of the recurrent parent for use in new hybrids andbreeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of a maize inbred line of interest, comprising thesteps of crossing a plant of a maize inbred line of interest with adonor plant comprising a mutant gene or transgene conferring a desiredtrait (i.e., a modulation in the expression of a ZmSNAC polynucleotideor any plant phenotype resulting from the modulated ZmSNAC expressionlevel such as those phenotypes discussed elsewhere herein, includingimproved abiotic stress tolerance); selecting an F1 progeny plantcomprising the mutant gene or transgene conferring the desired trait andbackcrossing the selected F1 progeny plant to a plant of the maizeinbred line of interest. This method may further comprise the step ofobtaining a molecular marker profile of the maize inbred line ofinterest and using the molecular marker profile to select for a progenyplant with the desired trait and the molecular marker profile of theinbred line of interest. In the same manner, this method may be used toproduce F1 hybrid seed by adding a final step of crossing the desiredtrait conversion of the maize inbred line of interest with a differentmaize plant to make F1 hybrid maize seed comprising a mutant gene ortransgene conferring the desired trait.

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. The method entails individual plantscross pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny,selfed progeny and toperossing. The selected progeny arecross-pollinated with each other to form progeny for another population.This population is planted and again superior plants are selected tocross pollinate with each other. Recurrent selection is a cyclicalprocess and therefore can be repeated as many times as desired. Theobjective of recurrent selection is to improve the traits of apopulation. The improved population can then be used as a source ofbreeding material to obtain inbred lines to be used in hybrids or usedas parents for a synthetic cultivar. A synthetic cultivar is theresultant progeny formed by the intercrossing of several selectedinbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation breeding is one of many methods that could be used to introducenew traits into an elite line. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation such as X-rays, Gamma rays (e.g.,cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14) or ultravioletradiation (preferably from 2500 to 2900 nm) or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development,” Fehr, 1993Macmillan Publishing Company, the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of elite lines that comprisessuch mutations.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplant species of interest include, but are not limited to, corn or maize(Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats (Avena sativa), barley (Hordeum vulgare),vegetables, ornamentals and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.) and members of the genus Cucumis such ascucumber (C. sativus), cantaloupe (C. cantalupensis) and musk melon (C.melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tu/ipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima) and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta) and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea) and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, corn and soybean plantsare optimal and in yet other embodiments corn plants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as maize, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

Typically, an intermediate host cell will be used in the practice ofthis invention to increase the copy number of the cloning vector. Withan increased copy number, the vector containing the nucleic acid ofinterest can be isolated in significant quantities for introduction intothe desired plant cells. In one embodiment, plant promoters that do notcause expression of the polypeptide in bacteria are employed.

Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res.8:4057) and the lambda derived P L promoter and N-gene ribosome bindingsite (Shimatake, et al., (1981) Nature 292:128). The inclusion ofselection markers in DNA vectors transfected in E coli. is also useful.Examples of such markers include genes specifying resistance toampicillin, tetracycline or chloramphenicol.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., (1983) Gene22:229-235); Mosbach, et al., (1983) Nature 302:543-545).

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells are known to those of skill in the art.As explained briefly below, a polynucleotide of the present inventioncan be expressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous polynucleotides in yeast is well known(Sherman, et al., (1982) Methods in Yeast Genetics, Cold Spring HarborLaboratory). Two widely utilized yeasts for production of eukaryoticproteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors,strains and protocols for expression in Saccharomyces and Pichia areknown in the art and available from commercial suppliers (e.g.,Invitrogen). Suitable vectors usually have expression control sequences,such as promoters, including 3-phosphoglycerate kinase or alcoholoxidase and an origin of replication, termination sequences and the likeas desired. A protein of the present invention, once expressed, can beisolated from yeast by lysing the cells and applying standard proteinisolation techniques to the lists. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay or other standard immunoassay techniques.

The sequences of the present invention can also be ligated to variousexpression vectors for use in transfecting cell cultures of, forinstance, mammalian, insect or plant origin. As with yeast, when higheranimal or plant host cells are employed, polyadenylation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenylationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript may also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., (1983)J. Virol. 45:773-781). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo,(1985) DNA Cloning Vol. II a Practical Approach, D. M. Glover, Ed., IRLPress, Arlington, Va., pp. 213-238).

II. Modulating the Level and/or Activity of a ZmSNAC Polypeptide

A method for modulating the level and/or activity of the polypeptide ofthe present invention in a plant is provided. In general, the leveland/or activity of the ZmSNAC polypeptide is increased or reduced by atleast 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more,relative to a native control plant, plant part or cell which does notcomprise the introduced sequence. Modulation of the level and/oractivity may occur at one or more stages of development. In specificembodiments, the polypeptides of the present invention are modulated inmonocots, such as maize.

The expression level of the ZmSNAC polypeptide may be measured directly,for example, by assaying for the level of the ZmSNAC polypeptide in theplant or indirectly, for example by measuring DNA binding activityand/or transgenic activation of gene expression in vitro or in vivo.

In specific embodiments, the polypeptide or the polynucleotide of theinvention is introduced into the plant cell. Subsequently, a plant cellhaving the introduced sequence of the invention is selected usingmethods known to those of skill in the art such as, but not limited to,Southern blot analysis, DNA sequencing, PCR analysis or phenotypicanalysis. A plant or plant part altered or modified by the foregoingembodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of polypeptidesof the present invention in the plant. Plant forming conditions are wellknown in the art and discussed briefly elsewhere herein.

It is also recognized that the level and/or activity of the polypeptidemay be modulated by employing a polynucleotide that is not capable ofdirecting, in a transformed plant, the expression of a protein or RNA.For example, the polynucleotides of the invention may be used to designpolynucleotide constructs that can be employed in methods for alteringor mutating a genomic nucleotide sequence in an organism. Suchpolynucleotide constructs include, but are not limited to, RNA:DNAvectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such nucleotideconstructs and methods of use are known in the art. See, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984, allof which are herein incorporated by reference. See also, WO 98/49350, WO99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad. Sci.USA 96:8774-8778, herein incorporated by reference.

It is therefore recognized that methods of the present invention do notdepend on the incorporation of the entire polynucleotide into thegenome, only that the plant or cell is altered as a result of theintroduction of the polynucleotide into a cell. In one embodiment of theinvention, the genome may be altered following the introduction of apolynucleotide into a cell. For example, the polynucleotide, or any partthereof, may incorporate into the genome of the plant. Alterations tothe genome include, but are not limited to, additions, deletions andsubstitutions of nucleotides into the genome. While the methods of thepresent invention do not depend on additions, deletions andsubstitutions of any particular number of nucleotides, it is recognizedthat such additions, deletions or substitutions comprise at least onenucleotide.

It is further recognized that modulating the level and/or activity ofthe ZmSNAC sequence can be manipulated so as to occur only duringcertain developmental stages. Control of ZmSNAC expression can beobtained via the use of inducible promoters, tissue-preferred promotersor promoters active exclusively or preferentially at one or moredevelopmental stages. Alternatively, the gene could be inverted ordeleted using site-specific recombinases, transposons or recombinationsystems.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been effected as to a gene of interest or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control cell” or “control plant cell” provides a reference point formeasuring changes in phenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

In the present case, for example, changes in ZmSNAC transcription factorlevels or activity could be measured by comparing performance of asubject plant or plant cell to a control plant or plant cell underdrought or other abiotic stress conditions. In addition, changes in geneexpression induced by ZmSNAC modulation could be assayed usinggene-expression profiling technologies.

In certain embodiments the nucleic acid constructs of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; for example, the constructs and methods of the inventioncould be used in combination with other genes that confer tolerance todrought or other abiotic stresses, such as salt or heat stress, as wellas with genes designed to enhance yield. Other combinations may bedesigned to produce plants with a variety of desired traits, such asthose described elsewhere herein.

A. Increasing the Activity and/or Level of a ZmSNAC Polypeptide

Methods are provided to increase the activity and/or level of a ZmSNACpolypeptide of the invention. An increase in the level of concentrationand/or activity of a ZmSNAC polypeptide of the invention can be achievedby providing to the plant a ZmSNAC polypeptide. As discussed elsewhereherein, many methods are known in the art for providing a polypeptide toa plant including, but not limited to, direct introduction of thepolypeptide into the plant and introducing into the plant (transientlyor stably) a polynucleotide construct encoding a polypeptide having SNACtranscription factor activity. Thus, the level and/or activity of aZmSNAC polypeptide may be increased by altering the gene encoding theZmSNAC polypeptide or its promoter. It is also recognized that themethods of the invention may employ a polynucleotide that is not capableof directing, in the transformed plant, the expression of a protein orRNA. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,PCT/US93/03868. Plants are provided which carry mutations affecting oneor more ZmSNAC genes, wherein the mutations increase expression of atleast one ZmSNAC gene or increase the level or activity of at least oneencoded ZmSNAC polypeptide. As described elsewhere herein, methods toassay for an increase in level or activity of a ZmSNAC polypeptide areknown.

B. Reducing the Activity and/or Level of a ZmNAC Polypeptide

Methods are provided to reduce or eliminate the activity and/or level(concentration) of a ZmSNAC polypeptide by transforming a plant cellwith an expression cassette that expresses a polynucleotide thatinhibits the expression of the ZmSNAC polypeptide. The polynucleotidemay inhibit the expression of a ZmSNAC polypeptide directly, bypreventing translation of the ZmSNAC polypeptide messenger RNA, orindirectly, by encoding a molecule that inhibits the transcription ortranslation of a gene encoding a ZmSNAC polypeptide. Methods forinhibiting or eliminating the expression of a gene in a plant are wellknown in the art and any such method may be used in the presentinvention to inhibit the expression of the ZmSNAC polypeptides.

In accordance with the present invention, the expression of a ZmSNACpolypeptide is inhibited if the level of the ZmSNAC polypeptide isstatistically lower than the level of the same ZmSNAC polypeptide in aplant that has not been genetically modified to inhibit the expressionof that ZmSNAC polypeptide. In particular embodiments of the invention,the level or activity of the ZmSNAC polypeptide in a modified plantaccording to the invention is less than 95%, less than 90%, less than80%, less than 70%, less than 60%, less than 50%, less than 40%, lessthan 30%, less than 20%, less than 10% or less than 5% of the level oractivity of the same ZmSNAC polypeptide in a control plant. Theexpression level of the ZmSNAC polypeptide may be measured directly, forexample, by assaying for the level of the ZmSNAC polypeptide expressedin the cell or plant, or indirectly, for example, by measuring DNAbinding activity or protein interaction in the cell or plant.

In other embodiments of the invention, the activity of one or moreZmSNAC polypeptides is reduced or eliminated by transforming a plantcell with an expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of one or more ZmSNACpolypeptides. The activity of a ZmSNAC polypeptide is inhibitedaccording to the present invention if the transcription factor activityof the ZmSNAC polypeptide is statistically lower than the correspondingactivity of the same ZmSNAC polypeptide in a control plant or cell. Inparticular embodiments of the invention, the activity of the ZmSNACpolypeptide in a modified plant according to the invention is less than95%, less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10% orless than 5% of the activity of the same ZmSNAC polypeptide in a controlplant or cell. The activity of a ZmSNAC polypeptide is “eliminated”according to the invention when it is not detectable by the assaymethods described elsewhere herein.

In other embodiments, the activity of a ZmSNAC polypeptide may bereduced or eliminated by disrupting the gene encoding the ZmSNACpolypeptide. The invention encompasses mutagenized plants that carrymutations in ZmSNAC genes, where the mutations reduce expression of theZmSNAC gene or inhibit the activity of the encoded ZmSNAC polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of aZmSNAC polypeptide. More than one method may be used to reduce theactivity of a single ZmSNAC polypeptide. In addition, combinations ofmethods may be employed to reduce or eliminate the activity of two ormore different ZmSNAC polypeptides.

Non-limiting examples of methods of reducing or eliminating theexpression of a ZmSNAC polypeptide are given below.

1. Polynucleotide-Based Methods

In some embodiments of the present invention, a plant cell istransformed with an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of a ZmSNAC sequence. Theterm “expression” as used herein refers to the biosynthesis of a geneproduct, including the transcription and/or translation of said geneproduct. For example, for the purposes of the present invention, anexpression cassette capable of expressing a polynucleotide that inhibitsthe expression of at least one ZmSNAC sequence is an expression cassettecapable of producing an RNA molecule that inhibits the transcriptionand/or translation of at least one ZmSNAC polypeptide. The “expression”or “production” of a protein or polypeptide from a DNA molecule refersto the transcription and translation of the coding sequence to producethe protein or polypeptide, while the “expression” or “production” of aprotein or polypeptide from an RNA molecule refers to the translation ofthe RNA coding sequence to produce the protein or polypeptide.

Examples of polynucleotides that inhibit the expression of a ZmSNACsequence are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of aZmSNAC polypeptide may be obtained by sense suppression orcosuppression. For cosuppression, an expression cassette is designed toexpress an RNA molecule corresponding to all or part of a messenger RNAencoding a ZmSNAC polypeptide in the “sense” orientation. Overexpression of the RNA molecule can result in reduced expression of thenative gene. Accordingly, multiple plant lines transformed with thecosuppression expression cassette are screened to identify those thatshow the greatest inhibition of ZmSNAC polypeptide expression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the ZmSNAC polypeptide, all or part of the 5′and/or 3′ untranslated region of a ZmSNAC polypeptide transcript or allor part of both the coding sequence and the untranslated regions of atranscript encoding a ZmSNAC polypeptide. In some embodiments where thepolynucleotide comprises all or part of the coding region for the ZmSNACpolypeptide, the expression cassette is designed to eliminate the startcodon of the polynucleotide so that no protein product will betranscribed.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001)Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731;Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos.5,034,323, 5,283,184 and 5,942,657, each of which is herein incorporatedby reference. The efficiency of cosuppression may be increased byincluding a poly-dT region in the expression cassette at a position 3′to the sense sequence and 5′ of the polyadenylation signal. See, USPatent Application Publication Number 2002/0048814, herein incorporatedby reference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323, herein incorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe ZmSNAC polypeptide may be obtained by antisense suppression. Forantisense suppression, the expression cassette is designed to express anRNA molecule complementary to all or part of a messenger RNA encodingthe ZmSNAC polypeptide. Overexpression of the antisense RNA molecule canresult in reduced expression of the native gene. Accordingly, multipleplant lines transformed with the antisense suppression expressioncassette are screened to identify those that show the desired inhibitionof ZmSNAC polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the ZmSNACpolypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the ZmSNAC polypeptide transcript or all or partof the complement of both the coding sequence and the untranslatedregions of a transcript encoding the ZmSNAC polypeptide. In addition,the antisense polynucleotide may be fully complementary (i.e., 100%identical to the complement of the target sequence) or partiallycomplementary (i.e., less than 100% identical to the complement of thetarget sequence) to the target sequence. Antisense suppression may beused to inhibit the expression of multiple proteins in the same plant.See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of theantisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may beused. Methods for using antisense suppression to inhibit the expressionof endogenous genes in plants are described, for example, in Liu, etal., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829and 5,942,657, each of which is herein incorporated by reference.Efficiency of antisense suppression may be increased by including apoly-dT region in the expression cassette at a position 3′ to theantisense sequence and 5′ of the polyadenylation signal. See, US PatentApplication Publication Number 2002/0048814, herein incorporated byreference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of aZmSNAC polypeptide may be obtained by double-stranded RNA (dsRNA)interference. For dsRNA interference, a sense RNA molecule like thatdescribed above for cosuppression and an antisense RNA molecule that isfully or partially complementary to the sense RNA molecule are expressedin the same cell, resulting in inhibition of the expression of thecorresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe desired level of inhibition of ZmSNAC polypeptide expression.Methods for using dsRNA interference to inhibit the expression ofendogenous plant genes are described in Waterhouse, et al., (1998) Proc.Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol.129:1732-1743 and WO 99/49029, WO 99/53050, WO 99/61631 and WO 00/49035,each of which is herein incorporated by reference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or more ZmSNAC polypeptides may be obtained by hairpin RNA (hpRNA)interference or intron-containing hairpin RNA (ihpRNA) interference.These methods are highly efficient at inhibiting the expression ofendogenous genes. See, Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoded by the gene whoseexpression is to be inhibited and an antisense sequence that is fully orpartially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouseand Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNAinterference to inhibit or silence the expression of genes aredescribed, for example, in Chuang and Meyerowitz, (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US PatentApplication Publication Number 2003/0175965, each of which is hereinincorporated by reference. A transient assay for the efficiency of hpRNAconstructs to silence gene expression in vivo has been described byPanstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, hereinincorporated by reference.

Alternatively, the base-paired stem region may correspond to a portionof a promoter sequence controlling expression of the gene to beinhibited. Transcriptional gene silencing (TGS) may be accomplishedthrough use of hpRNA constructs wherein the inverted repeat of thehairpin shares sequence identity with the promoter region drivingexpression of a gene to be silenced. See, for example, US PatentApplication Publication 2005/0246796. Processing of the hpRNA into shortRNAs which can interact with the homologous promoter region may triggerdegradation or methylation to result in silencing (Aufsatz, et al.,(2002) PNAS 99(4):16499-16506; Mette, et al., (2000) EMBO J19(19):5194-5201). In other embodiments, the hairpin inverted repeat isbased on, and homologous to, the 3′ untranslated region of the gene tobe inhibited.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295and US Patent Application Publication Number 2003/0180945, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for ZmSNAC polypeptide). Methods ofusing amplicons to inhibit the expression of endogenous plant genes aredescribed, for example, in Angell and Baulcombe, (1997) EMBO J.16:3675-3684, Angell and Baulcombe, (1999) Plant J. 20:357-362 and U.S.Pat. No. 6,635,805, each of which is herein incorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of a ZmSNAC polypeptide. Thus, thepolynucleotide causes the degradation of the endogenous messenger RNA,resulting in reduced expression of the ZmSNAC polypeptide. This methodis described, for example, in U.S. Pat. No. 4,987,071, hereinincorporated by reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression ofone or more ZmSNAC polypeptides may be obtained by RNA interference byexpression of a gene encoding a micro RNA (miRNA). miRNAs are regulatoryagents consisting of about 22 ribonucleotides. miRNA are highlyefficient at inhibiting the expression of endogenous genes. See, forexample, Javier, et al., (2003) Nature 425:257-263, herein incorporatedby reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of ZmSNAC polypeptide expression, the22-nucleotide sequence is selected from a ZmSNAC polypeptide transcriptsequence and contains 22 nucleotides encoding said ZmSNAC polypeptidesequence in sense orientation and 21 nucleotides of a correspondingantisense sequence that is complementary to the sense sequence. miRNAmolecules are highly efficient at inhibiting the expression ofendogenous genes and the RNA interference they induce is inherited bysubsequent generations of plants.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a ZmSNAC polypeptide, resulting in reducedexpression of the gene. In particular embodiments, the zinc fingerprotein binds to a regulatory region of a ZmSNAC polypeptide gene. Inother embodiments, the zinc finger protein binds to a messenger RNAencoding a ZmSNAC polypeptide and prevents its translation. Methods ofselecting sites for targeting by zinc finger proteins have beendescribed, for example, in U.S. Pat. No. 6,453,242 and methods for usingzinc finger proteins to inhibit the expression of genes in plants aredescribed, for example, in US Patent Application Publication Number2003/0037355, each of which is herein incorporated by reference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one ZmSNAC polypeptide and reduces theactivity of the ZmSNAC polypeptide. In another embodiment, the bindingof the antibody results in increased turnover of the antibody-ZmSNACpolypeptide complex by cellular quality control mechanisms. Theexpression of antibodies in plant cells and the inhibition of molecularpathways by expression and binding of antibodies to proteins in plantcells are well known in the art. See, for example, Conrad and Sonnewald,(2003) Nature Biotech. 21:35-36, incorporated herein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of a ZmSNACpolypeptide is reduced or eliminated by disrupting the gene encoding theZmSNAC polypeptide. The gene encoding the ZmSNAC polypeptide may bedisrupted by any method known in the art. For example, in oneembodiment, the gene is disrupted by transposon tagging. In anotherembodiment, the gene is disrupted by mutagenizing plants using random ortargeted mutagenesis, and selecting for plants that have reduced ZmSNACactivity. ZmSNAC down-regulated plants could be assayed for a decreasein expression of either ZmSNAC itself or of genes regulated by it. Inaddition, downregulation of ZmSNAC should have a measureable phenotyperelated to drought and yield.

i. Transposon Tagging

In one embodiment of the invention, the expression of one or more ZmSNACpolypeptides is reduced or eliminated by inserting a transposon within aregulatory region or coding region of the gene encoding the ZmSNACpolypeptide. A transposon may be inserted within an exon, intron, 5′ or3′ untranslated sequence, a promoter, or any other regulatory sequenceof a ZmSNAC polynucleotide. Methods for the transposon tagging ofspecific genes in plants are well known in the art. See, for example,Maes, et al., (1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti,(1999) FEMS Microbiol. Lett. 179:53-59; Meissner, et al., (2000) PlantJ. 22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot,(2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000) NucleicAcids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics153:1919-1928). In addition, the TUSC process for selecting Muinsertions in selected genes has been described in Bensen, et al.,(1995) Plant Cell 7:75-84; Mena, et al., (1996) Science 274:1537-1540;and U.S. Pat. No. 5,962,764, each of which is herein incorporated byreference.

ii. Mutant Plants with Reduced Activity

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics154:421-436, each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products, is also applicable to the instant invention. See,McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, hereinincorporated by reference.

Mutations that impact gene expression or that interfere with thefunction of the encoded protein are well known in the art. Insertionalmutations in gene exons usually result in null mutants. Mutations inconserved residues are particularly effective in inhibiting the activityof the encoded protein. Conserved residues of NAC polypeptides have beendescribed (Ernst, et al., (2004) EMBO Reports 5(3):297-303) and providea basis for designing mutations with the goal of eliminating the ZmSNACtranscription factor activity. See, for example, FIG. 1. Such mutantscan be isolated according to well-known procedures, and mutations indifferent ZmSNAC loci can be stacked by genetic crossing. See, forexample, Gruis, et al., (2002) Plant Cell 14:2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba, et al., (2003) PlantCell 15:1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more ZmSNAC polypeptides. Examples of othermethods for altering or mutating a genomic nucleotide sequence in aplant are known in the art and include, but are not limited to, the useof RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors,mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such vectors andmethods of use are known in the art. See, for example, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984,each of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778, each of which is herein incorporatedby reference.

III. Modulating ZmSNAC Transcription Factor Level and/or Activity

“Modulating ZmSNAC transcription factor level and/or activity” includesany statistically significant decrease or increase in level and/oractivity of said factor in the plant when compared to a control plant.The modulated level and/or activity of the ZmSNAC transcription factorcan comprise either an increase or a decrease of about 0.1%, 0.5%, 1%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more when compared to a control. It isfurther recognized that the modulation of the ZmSNAC level/activity neednot be an overall increase/decrease in ZmSNAC level and/or activity, butmay comprise a change in tissue distribution of the transcription factoractivity. Moreover, the modulation of the transcription factorlevel/activity need not be an overall increase/decrease, but alsoincludes a change in the ratio of activity of various transcriptionfactors.

Methods for assaying a modulation in transcription factor level and/oractivity are known in the art. As discussed elsewhere herein, modulationin ZmSNAC level and/or activity can further be detected by monitoringfor particular plant phenotypes. Because ZmSNAC genes regulate theexpression of other genes, modulation of a ZmSNAC gene should bemeasurable by changes in target gene expression.

In specific methods, the level and/or activity of a ZmSNAC transcriptionfactor in a plant is increased by increasing the level or activity ofthe ZmSNAC polypeptide in the plant. Methods for increasing the leveland/or activity of ZmSNAC polypeptides in a plant are discussedelsewhere herein. In certain embodiments, a ZmSNAC nucleotide sequenceencoding a ZmSNAC polypeptide can be provided by introducing into theplant a polynucleotide comprising a ZmSNAC nucleotide sequence of theinvention, expressing the ZmSNAC sequence and thereby increasing thelevel and/or activity of said transcription factor in the plant or plantpart when compared to a control. In some embodiments, the ZmSNACnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant.

In other methods, the level and/or activity of ZmSNAC transcriptionfactors in a plant is decreased by decreasing the level and/or activityof one or more of the ZmSNAC polypeptides in the plant. Such methods aredisclosed in detail elsewhere herein. In one such method, a ZmSNACnucleotide sequence is introduced into the plant and expression of theZmSNAC nucleotide sequence decreases the level and/or activity of theZmSNAC polypeptide in the plant or plant part when compared to a controlplant or plant part. In certain embodiments, the nucleotide constructintroduced into the plant is stably incorporated into the genome of theplant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate the level/activity of a ZmSNAC transcription factorin the plant. Exemplary promoters for this embodiment have beendisclosed elsewhere herein.

Accordingly, the present invention further provides plants having amodulated level/activity of a ZmSNAC transcription factor when comparedto said level/activity in a control plant. In one embodiment, the plantof the invention has an increased level/activity of the ZmSNACpolypeptide of the invention. In other embodiments, the plant of theinvention has a reduced or eliminated level of the ZmSNAC polypeptide ofthe invention. In certain embodiments, such plants have stablyincorporated into their genome a nucleic acid molecule comprising aZmSNAC nucleotide sequence of the invention operably linked to apromoter that drives expression in the plant cell.

A. Modulating Root Development

Modulation of the level/activity of a ZmSNAC transcription factor mayresult in modulated root development when compared to a control plant.Such alterations in root development include, but are not limited to,alterations in the size or growth rate of the primary root, the freshroot weight, the extent of lateral and adventitious root formation, thevasculature system, meristem development and radial expansion. Methodsof measuring such developmental alterations in the root system are knownin the art. See, for example, US Patent Application Publication Number2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both ofwhich are herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate root development in the plant. Exemplary promotersfor this embodiment include constitutive promoters and root-preferredpromoters. Exemplary root-preferred promoters have been disclosedelsewhere herein.

Modulated root development may also impact the standability of a plant.The term “resistance to lodging” or “standability” refers to the abilityof a plant to fix itself to the soil. For plants with an erect orsemi-erect growth habit, this term also refers to the ability tomaintain an upright position under adverse environmental conditions.This trait relates to the size, depth and morphology of the root system.In addition, stimulating root growth and increasing root mass atappropriate developmental stages also finds use in promoting in vitropropagation of explants.

Increased root biomass and/or altered root architecture may also finduse in improving nitrogen-use efficiency of the plant. Such improvedefficiency may lead to, for example, an increase in plant biomass and/orseed yield at an existing level of available nitrogen or maintenance ofplant biomass and/or seed yield when available nitrogen is limited.Thus, agronomic and/or environmental benefits may ensue.

Furthermore, higher root biomass production has an indirect effect onproduction of compounds produced by root cells or transgenic root cellsor cell cultures of said transgenic root cells. One example of aninteresting compound produced in root cultures is shikonin, the yield ofwhich can be advantageously enhanced by said methods.

B. Modulating Shoot and Leaf Development

Methods are also provided for modulating vegetative tissue growth inplants. In one embodiment, shoot and/or leaf development in a plant ismodulated when compared to a control plant or plant part. Suchalterations in shoot and/or leaf development include, but are notlimited to, alterations in shoot meristem development, in leaf number,leaf size, leaf and stem vasculature, internode length and leafsenescence. As used herein, “leaf development” and “shoot development”encompasses all aspects of growth of the different parts that make upthe leaf system and the shoot system, respectively, at different stagesof their development, both in monocotyledonous and dicotyledonousplants. Methods for measuring such developmental alterations in theshoot and leaf system are known in the art. See, for example, Werner, etal., (2001) PNAS 98:10487-10492 and US Patent Application PublicationNumber 2003/0074698, each of which is herein incorporated by reference.

The method for modulating shoot and/or leaf development in a plantcomprises modulating the activity and/or level of a ZmSNAC polypeptideof the invention. In one embodiment, a ZmSNAC sequence of the inventionis provided. In other embodiments, the ZmSNAC nucleotide sequence can beprovided by introducing into the plant a polynucleotide comprising aZmSNAC nucleotide sequence of the invention, expressing the ZmSNACsequence and thereby modifying shoot and/or leaf development. In certainembodiments, the ZmSNAC nucleotide construct introduced into the plantis stably incorporated into the genome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate shoot and leaf development of the plant. Exemplarypromoters for this embodiment include constitutive promoters,shoot-preferred promoters, shoot meristem-preferred promoters,senescence-activated promoters, stress-induced promoters,nitrogen-induced promoters and leaf-preferred promoters. Exemplarypromoters have been disclosed elsewhere herein.

Decreasing ZmSNAC activity in a plant generally results in shorterinternodes and stunted growth. Thus, the methods of the invention finduse in producing dwarf plants. In addition, as discussed above,modulation of ZmSNAC activity in the plant may modulate both root andshoot growth. Thus, the present invention further provides methods foraltering the root/shoot ratio.

It is further recognized that increasing seed size and/or weight can beaccompanied by an increase in the rate of growth of seedlings or anincrease in vigor. In addition, modulating the plant's tolerance tostress, as discussed elsewhere herein, along with modulation of root,shoot and leaf development, can increase plant yield and vigor. As usedherein, the term “vigor” refers to the relative health, productivity andrate of growth of the plant and/or of certain plant parts and may bereflected in various developmental attributes, including, but notlimited to, concentration of chlorophyll, photosynthetic rate, totalbiomass, root biomass, grain quality and/or grain yield. In Zea mays inparticular, vigor may also be reflected in ear growth rate, ear sizeand/or rate or degree of silk exsertion. Vigor may relate to the abilityof a plant to grow rapidly during early development and to thesuccessful establishment of a well-developed root system and awell-developed photosynthetic apparatus. Vigor may be determined withreference to different genotypes under similar environmental conditionsor with reference to the same or different genotypes under differentenvironmental conditions.

Accordingly, the present invention further provides plants havingmodulated shoot and/or leaf development when compared to a controlplant. In some embodiments, the plant of the invention has an increasedlevel/activity of a ZmSNAC polypeptide of the invention. In otherembodiments, the plant of the invention has a decreased level/activityof the ZmSNAC polypeptide of the invention.

C. Modulating Reproductive Tissue Development

Methods for modulating reproductive tissue development are provided. Inone embodiment, methods are provided to modulate floral development in aplant. By “modulating floral development” is intended any alteration ina structure of a plant's reproductive tissue as compared to a controlplant or plant part. “Modulating floral development” further includesany alteration in the timing of the development of a plant'sreproductive tissue (i.e., delayed or accelerated floral development)when compared to a control plant or plant part. Macroscopic alterationsmay include changes in size, shape, number or location of reproductiveorgans, the developmental time period during which these structures formor the ability to maintain or proceed through the flowering process intimes of environmental stress. Microscopic alterations may includechanges to the types or shapes of cells that make up the reproductiveorgans.

The method for modulating floral development in a plant comprisesmodulating (either increasing or decreasing) the level and/or activityof the ZmSNAC polypeptide in a plant. In one method, a ZmSNAC sequenceof the invention is provided. A ZmSNAC nucleotide sequence can beprovided by introducing into the plant a polynucleotide comprising aZmSNAC nucleotide sequence of the invention, expressing the ZmSNACsequence and thereby modifying floral development. In some embodiments,the ZmSNAC nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate floral development in the plant. Exemplary promotersfor this embodiment include constitutive promoters, inducible promoters,shoot-preferred promoters and inflorescence-preferred promoters(including developing-female-inflorescence-preferred promoters),including those listed elsewhere herein.

Accordingly, the present invention further provides plants havingmodulated floral development when compared to the floral development ofa control plant. Compositions include plants with a modulatedlevel/activity of one or more ZmSNAC polypeptides of the invention andhaving an altered floral development, which may include the capacity tosustain normal reproductive development under abiotic stress conditions.

D. Modulating the Stress Tolerance of a Plant

Methods are provided for the use of the ZmSNAC sequences of theinvention to modify the tolerance of a plant to abiotic stress.Promoters that can be used in this method are described elsewhereherein, including low-level constitutive, stress-insensitive orinducible, particularly stress-inducible, promoters. Accordingly, in onemethod of the invention, a plant's tolerance to stress is increased ormaintained when compared to a control plant by introducing into theplant a polynucleotide comprising a ZmSNAC nucleotide sequence of theinvention, expressing the ZmSNAC sequence, and thereby increasing theplant's tolerance to stress. In certain embodiments, the ZmSNACnucleotide construct introduced into the plant is stably incorporatedinto the genome of the plant.

Increased growth of seedlings or early vigor is often associated with anincrease in stress tolerance. For example, faster development ofseedlings, including the root system of seedlings upon germination, iscritical for survival, particularly under adverse conditions such asdrought.

Methods are also provided to increase or maintain seed set duringabiotic stress episodes. During periods of stress (i.e., drought, salt,heavy metals, temperature, etc.) embryo development is often aborted. Inmaize, halted embryo development results in aborted kernels on the ear(Cheikh and Jones, (1994) Plant Physiol. 106:45-51; Dietrich, et al.,(1995) Plant Physiol Biochem 33:327-336). In soy, abortion of pods priorto seed maturation can reduce seed yield and is observed during bothoptimal and stress conditions. Preventing this seed loss will maintainyield. Accordingly, methods are provided to increase the stressresistance in a plant (e.g., during flowering and seed development).Increasing expression of the ZmSNAC sequence of the invention canmodulate floral development during periods of stress and thus methodsare provided to maintain or improve the flowering process in plantsunder stress. The method comprises increasing the level and/or activityof one or more of the ZmSNAC sequences of the invention. In one method,a ZmSNAC nucleotide sequence is introduced into the plant and the leveland/or activity of the ZmSNAC polypeptide is increased, therebymaintaining or improving the tolerance of the plant under stressconditions. In certain methods, the ZmSNAC nucleotide constructintroduced into the plant is stably incorporated into the genome of theplant.

Significant yield instability can occur as a result of unfavorableenvironments during the lag phase of seed development. During thisperiod, seeds undergo dramatic changes in ultra structure, biochemistryand sensitivity to environmental perturbation, yet demonstrate littlechange in dry mass accumulation. Two important events that occur duringthe lag phase are initiation and division of endosperm cells andamyloplasts (which are the sites for starch deposition). In crop speciessuch as maize, kernel sink capacity is principally a function of thenumber of endosperm cells and starch granules established during thefirst 6 to 12 days after pollination. The final number of endospermcells and amyloplasts formed is highly correlated with final kernelweight. (Capitanio, et al., (1983); Reddy and Daynard, (1983); Jones, etal., (1985) (1996); Engelen-Eigles, et al., (2000)).

In this embodiment, a variety of promoters could be used to direct theexpression of a sequence capable of increasing the level and/or activityof the ZmSNAC polypeptide, including but not limited to, constitutivepromoters, seed-preferred promoters, developing-seed promoters,meristem-preferred promoters, stress-induced promoters andinflorescence-preferred (such as developing female inflorescencepromoters). In one method, a promoter that is stress-inducible and isexpressed in a tissue of the developing seed during the lag phase ofdevelopment is used. By “lag phase” promoter is intended a promoter thatis active in the lag phase of seed development. A description of thisdevelopmental phase is found elsewhere herein. By“developing-seed-preferred” is intended a promoter that allows forenhanced ZmSNAC expression within a developing seed. Such promoters thatare stress insensitive and are expressed in a tissue of the developingseed during the lag phase of development are known in the art andinclude Zag2.1 (Theissen, et al., (1995) Gene 156:155-166, GenbankAccession Number X80206) and mzE40 (Zm40) (U.S. Pat. No. 6,403,862 andWO01/2178).

Methods to assay for an increase in seed set during abiotic stress areknown in the art. For example, plants having the increased ZmSNACactivity can be monitored under various stress conditions and comparedto control plants. For instance, the plant having the increased ZmSNACtranscription factor activity can be subjected to various degrees ofstress during flowering and seed set. Under identical conditions, thegenetically modified plant having the increased ZmSNAC transcriptionfactor activity will have a higher number of developing pods and/orseeds than a control plant.

Accordingly, the present invention further provides plants havingincreased yield or a maintained yield and/or an increased or maintainedflowering process during periods of abiotic stress (for example,drought, salt, heavy metals, temperature extremes). In some embodiments,the plants having an increased or maintained yield during abiotic stresshave an increased level/activity of the ZmSNAC polypeptide of theinvention. In some embodiments, the plant comprises a ZmSNAC nucleotidesequence of the invention operably linked to a promoter that drivesexpression in the plant cell. In some embodiments, such plants havestably incorporated into their genome a nucleic acid molecule comprisinga ZmSNAC nucleotide sequence of the invention operably linked to apromoter that drives expression in the plant cell.

IV. Antibody Creation and Use

Antibodies can be raised to a protein of the present invention,including variants and fragments thereof, in both theirnaturally-occurring and recombinant forms. Many methods of makingantibodies are known to persons of skill. A variety of analytic methodsare available to generate a hydrophilicity profile of a protein of thepresent invention. Such methods can be used to guide the artisan in theselection of peptides of the present invention for use in the generationor selection of antibodies which are specifically reactive, underimmunogenic conditions, to a protein of the present invention. See,e.g., Janin, (1979) Nature, 277:491-492; Wolfenden, et al., (1981)Biochemistry 208:49-855; Kyte and Doolite, (1982) J. Mol Biol.157:105-132; Rose, et al., (1985) Science 229:834-838. The antibodiescan be used to screen expression libraries for particular expressionproducts such as normal or abnormal protein or altered levels of thesame, which may be useful for detecting or diagnosing various conditionsrelated to the presence of the respective antigens. Assays indicatinghigh levels of a ZmSNAC protein of the invention, for example, could beuseful in detecting plants or specific plant parts, with elevated ZmSNACtranscription factor levels. Usually the antibodies in such a procedureare labeled with a moiety which allows easy detection of presence ofantigen/antibody binding.

The ZmSNAC1 regulatory element will be operably linked to a sequence ofinterest, which will provide for modification of the phenotype of theplant. Such modification includes modulating the production of anendogenous product, as to amount, relative distribution, or the like orfor providing a novel function or expression product. For example, sucha promoter is useful for modulation of expression of sequences encodingstress-responsive proteins, including other transcription factors.Additionally, linking a stress-induced promoter with a marker, and, inparticular, a visual marker, may be useful in tracking the expression ofa linked gene of interest. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, (1987) Plant Mol. Biol. Rep. 5.287 and U.S. Pat. No.5,599,670; Teeri, et al., (1989) EMBO J. 8:343, Koncz, et al., (1987)Proc. Natl. Acad. Sci. U.S.A. 84:131, De Block, et al., (1984) EMBO J.3:1681. Another approach to the identification of relatively raretransformation events has been use of a gene that encodes a dominantconstitutive regulator of the Zea mays anthocyanin pigmentation pathway.Ludwig, et al., (1990) Science 247:449.

A method for expressing an isolated nucleotide sequence in a plant usingthe regulatory sequences disclosed herein is provided. The methodcomprises transforming a plant cell with a transformation vector thatcomprises an isolated nucleotide sequence operably linked to a plantregulatory sequence of the present invention and regenerating a stablytransformed plant from the transformed plant cell. In this manner, theregulatory sequences are useful for controlling the expression ofendogenous as well as exogenous products in a stress-induced manner.

Frequently it is desirable to have preferential expression of a DNAsequence in a tissue of an organism or under certain environmentalconditions. For example, increased resistance of a plant to insectattack might be accomplished by genetic manipulation of the plant'sgenome to comprise a tissue-specific promoter operably linked to aheterologous insecticide gene such that the insect-deterring substancesare specifically expressed in the susceptible plant tissues. Increasedtolerance to abiotic stress might be accomplished by geneticmanipulation of the plant's genome to comprise a stress-induced promoteroperably linked to a heterologous gene encoding a biosynthetic orregulatory gene for a plant hormone such that the hormone isspecifically synthesized or its synthesis is regulated under the stressconditions. Preferential expression of the heterologous nucleotidesequence in the appropriate tissue or under the appropriate conditionsreduces the drain on the plant's resources that occurs when aconstitutive promoter initiates transcription of a heterologousnucleotide sequence throughout the cells of the plant and/or under allconditions.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype.For example, a hairpin configuration comprising all or a portion of aZmSNAC1 promoter may be used to downregulate the nativestress-responsive ZmSNAC1.

By “regulatory element” is intended sequences responsible for expressionof the associated coding sequence including, but not limited to,promoters, terminators, enhancers, introns and the like.

By “terminator” is intended a regulatory region of DNA that causes RNApolymerase to disassociate from DNA, causing termination oftranscription.

By “promoter” is intended a regulatory region of DNA capable ofregulating the transcription of a sequence linked thereto. It usuallycomprises a TATA box capable of directing RNA polymerase II to initiateRNA synthesis at the appropriate transcription initiation site for aparticular coding sequence.

A promoter may additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate and further include elements which impact spatial and temporalexpression of the linked nucleotide sequence. It is recognized thathaving identified the nucleotide sequences for the promoter regiondisclosed herein, it is within the state of the art to isolate andidentify further regulatory elements in the 5′ region upstream from theparticular promoter region identified herein. Thus the promoter regiondisclosed herein may comprise upstream regulatory elements such as thoseresponsible for tissue and temporal expression of the coding sequenceand may include enhancers, the DNA response element for atranscriptional regulatory protein, ribosomal binding sites,transcriptional start and stop sequences, translational start and stopsequences, activator sequence and the like.

In the same manner, the promoter elements which enable expression understress conditions can be identified, isolated and used with other corepromoters. By core promoter is meant the minimal sequence required toinitiate transcription, such as the sequence called the TATA box whichis common to promoters in genes encoding proteins. Thus the upstreampromoter of ZmSNAC1 can optionally be used in conjunction with its ownor core promoters from other sources. The promoter may be native ornon-native to the cell in which it is found.

The isolated promoter sequence of the present invention can be modifiedto provide for a range of expression levels of the isolated nucleotidesequence. Less than the entire promoter region can be utilized and theability to drive stress-induced expression retained. It is recognizedthat expression levels of mRNA can be modulated with specific deletionsof portions of the promoter sequence. Thus, the promoter can be modifiedto be a weak or strong promoter. Generally, by “weak promoter” isintended a promoter that drives expression of a coding sequence at a lowlevel. By “low level” is intended levels of about 1/10,000 transcriptsto about 1/100,000 transcripts to about 1/500,000 transcripts.Conversely, a strong promoter drives expression of a coding sequence ata high level, or at about 1/10 transcripts to about 1/100 transcripts toabout 1/1,000 transcripts. Generally, at least about 20 nucleotides ofan isolated promoter sequence will be used to drive expression of anucleotide sequence.

It is recognized that to increase transcription levels, enhancers can beutilized in combination with the promoter regions of the invention.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are known in the art and include theSV40 enhancer region, the 35S enhancer element and the like.

The promoter of the present invention can be isolated from the 5′ regionof its native coding region or 5′ untranslated region (5′ UTR). Likewisethe terminator can be isolated from the 3′ region flanking itsrespective stop codon. The term “isolated” refers to material, such as anucleic acid or protein, (1) which is substantially or essentially freefrom components which normally accompany or interact with the materialas found in its naturally occurring environment or (2) if the materialis in its natural environment, the material has been altered bydeliberate human intervention to a composition and/or placed at a locusin a cell other than the locus native to the material. Methods forisolation of promoter regions are well known in the art.

The Zea mays SNAC1 promoter is set forth in SEQ ID NO: 11 and is 1625nucleotides in length.

The promoter regions from genes homologous to ZmSNAC1 may be isolatedfrom any plant, including, but not limited to corn (Zea mays), canola(Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), millet (Panicumspp.), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), oats (Avena sativa), barley (Hordeum vulgare),vegetables, ornamentals and conifers. Preferably, plants include corn,soybean, sunflower, safflower, canola, wheat, barley, rye, alfalfa andsorghum.

Promoter sequences from other plants may be isolated according towell-known techniques based on sequence homology. In these techniques,all or part of the known coding sequence is used as a probe whichselectively hybridizes to other sequences present in a population ofcloned genomic DNA fragments (i.e., genomic libraries) from a chosenorganism. Methods are readily available in the art for the hybridizationof nucleic acid sequences. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays”, Elsevier, New York (1993); and CurrentProtocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., GreenePublishing and Wiley-Interscience, New York (1995).

“Functional variants” of the regulatory sequences are also encompassedby the compositions of the present invention. Functional variantsinclude, for example, the native regulatory sequences of the inventionhaving one or more nucleotide substitutions, deletions or insertions andwhich drive expression of an operably-linked sequence under conditionssimilar to those under which the native promoter is active. Functionalvariants of the invention may be created by site-directed mutagenesis,induced mutation or may occur as allelic variants (polymorphisms).

As used herein, a “functional fragment” is a truncated regulatorysequence formed by one or more deletions from a larger regulatoryelement. For example, the 5′ portion of a promoter up to the TATA boxnear the transcription start site can be deleted without abolishingpromoter activity, as described by Opsahl-Sorteberg, et al., (2004) Gene341:49-58. Such fragments should retain promoter activity, particularlythe ability to drive stress-induced expression. Activity can be measuredby Northern blot analysis, reporter activity measurements when usingtranscriptional fusions, and the like. See, for example, Sambrook, etal., (1989) Molecular Cloning: A Laboratory Manual (2nd ed. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.), herein incorporated byreference.

Functional fragments can be obtained by use of restriction enzymes tocleave the naturally occurring regulatory element nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring DNA sequence or can be obtained through the use ofPCR technology. See particularly, Mullis, et al., (1987) MethodsEnzymol. 155:335-350 and Erlich, ed. (1989) PCR Technology (StocktonPress, New York).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′,4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

The entire promoter sequence or portions thereof can be used as a probecapable of specifically hybridizing to corresponding promoter sequencesin genomic DNA. Alternatively, the probe represents a fragment of thecoding sequence natively associated with the promoter sequence. Toachieve specific hybridization under a variety of conditions, suchprobes include sequences that are unique and are preferably at leastabout 10 nucleotides in length, and most preferably at least about 20nucleotides in length. Such probes can be used to amplify correspondingsequences from a chosen organism by the well-known process of polymerasechain reaction (PCR). This technique can be used to isolate additionalpromoter sequences from a desired organism or as a diagnostic assay todetermine the presence of the promoter sequence in an organism. Examplesinclude hybridization screening of plated DNA libraries (either plaquesor colonies; see, e.g., Innis, et al., (1990) PCR Protocols, A Guide toMethods and Applications, eds., Academic Press).

The regulatory elements disclosed in the present invention, as well asvariants and fragments thereof, are useful in the genetic manipulationof any plant when operably linked with an isolated nucleotide sequenceof interest whose expression is to be controlled to achieve a desiredphenotypic response.

By “operably linked” is intended a functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. The expression cassette will include a regulatory sequence ofthe invention operably linked to at least one sequence of interest.

In one typical embodiment, in the context of an over expressioncassette, operably linked means that the nucleotide sequences beinglinked are contiguous and, where necessary to join two or more proteincoding regions, contiguous and in the same reading frame. In the casewhere an expression cassette contains two or more protein coding regionsjoined in a contiguous manner in the same reading frame, the encodedpolypeptide is herein defined as a “chimeric polypeptide” or a “fusionpolypeptide”. The cassette may additionally contain at least oneadditional coding sequence to be co-transformed into the organism.Alternatively, the additional coding sequence(s) can be provided onmultiple expression cassettes.

The regulatory elements of the invention can be operably linked to theisolated nucleotide sequence of interest in any of several ways known toone of skill in the art. The isolated nucleotide sequence of interestcan be inserted into a site within the genome which is 3′ to thepromoter of the invention using site specific integration as describedin U.S. Pat. No. 6,187,994, herein incorporated in its entirety byreference.

The regulatory elements of the invention can be operably linked inexpression cassettes along with isolated nucleotide sequences ofinterest for expression in the plant. Such an expression cassette isprovided with a plurality of restriction sites for insertion of thenucleotide sequence of interest under the transcriptional control of theregulatory elements.

The isolated nucleotides of interest expressed by the regulatoryelements of the invention can be used for directing expression of asequence in plant tissues. This can be achieved by increasing expressionof endogenous or exogenous products. Alternatively, the results can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors. This downregulation can be achieved through many different approaches known toone skilled in the art, including antisense, cosuppression, use ofhairpin formations or others and discussed infra. It is recognized thatthe regulatory elements may be used with their native or other codingsequences to increase or decrease expression of an operably linkedsequence in the transformed plant or seed.

General categories of genes of interest for the purposes of the presentinvention include for example, those genes involved in information, suchas zinc fingers; those involved in communication, such as kinases; andthose involved in housekeeping, such as heat shock proteins. Morespecific categories of transgenes include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance and grain characteristics. Still other categories oftransgenes include genes for inducing synthesis of exogenous productssuch as enzymes, cofactors and hormones from plants and other eukaryotesas well as prokaryotic organisms.

Modifications that affect grain traits include increasing the content ofoleic acid, or altering levels of saturated and unsaturated fatty acids.Likewise, the level of proteins, particularly modified proteins thatimprove the nutrient value of the plant, can be increased. This isachieved by the expression of such proteins having enhanced amino acidcontent.

Increasing the levels of lysine and sulfur-containing amino acids may bedesired as well as the modification of starch type and content in theseed. Hordothionin protein modifications are described in WO 9416078filed Apr. 10, 1997; WO 9638562 filed Mar. 26, 1997; WO 9638563 filedMar. 26, 1997 and U.S. Pat. No. 5,703,049 issued Dec. 30, 1997. Anotherexample is lysine and/or sulfur-rich root protein encoded by the soybean2S albumin described in WO 9735023 filed Mar. 20, 1996, and thechymotrypsin inhibitor from barley, Williamson, et al., (1987) Eur. J.Biochem. 165:99-106.

Agronomic traits can be improved by altering expression of genes that:affect the response of root, plant or seed growth and development duringenvironmental stress, Cheikh-N, et al., (1994) Plant Physiol.106(1):45-51 and genes controlling carbohydrate metabolism to reducekernel abortion in maize, Zinselmeier, et al., (1995) Plant Physiol.107(2):385-391.

It is recognized that any gene of interest, including the native codingsequence, can be operably linked to the regulatory elements of theinvention and expressed in the plant.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Maize Transformation

For example, immature maize embryos from greenhouse donor plants arebombarded with a plasmid containing a sequence, for example ZmSNAC1,operably linked to a constitutive promoter and further containing aselectable marker gene. Alternatively, the selectable marker gene isprovided on a separate plasmid. Transformation is performed as follows.Media recipes follow below.

The ears are husked and surface-sterilized in 30% Clorox® bleach plus0.5% Micro detergent for 20 minutes and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

A plasmid vector comprising the ZmSNAC sequence operably linked to aconstitutive promoter is made. This plasmid DNA plus plasmid DNAcontaining a selectable marker is precipitated onto 1.1 μm (averagediameter) tungsten pellets using a CaCl₂ precipitation procedure asfollows: 100 μl prepared tungsten particles in water; 10 μl (1 μg) DNAin Tris EDTA buffer (1 μg total DNA); 100 μl 2.5 M CaCl₂; and, 10 μl 0.1M spermidine.

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for the maintenance or increase of seedset during an abiotic stress episode.

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000X SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite® gelling agent (added after bringing to volumewith D-I H₂O) and 8.5 mg/l silver nitrate (added after sterilizing themedium and cooling to room temperature). Selection medium (560R)comprises 4.0 g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson'sVitamin Mix (1000X SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucroseand 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustmentto pH 5.8 with KOH); 3.0 g/l Gelrite® gelling agent (added afterbringing to volume with D-I H₂O) and 0.85 mg/l silver nitrate and 3.0mg/l bialaphos (both added after sterilizing the medium and cooling toroom temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite® gellingagent (added after bringing to volume with D-I H₂O) and 1.0 mg/lindoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing themedium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution(0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxineHCL and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1g/l myo-inositol and 40.0 g/l sucrose (brought to volume with polishedD-I H₂O after adjusting pH to 5.6) and 6 g/l Bacto™-agar solidifyingagent (added after bringing to volume with polished D-I H₂O), sterilizedand cooled to 60° C.

Example 2 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing the ZmSNAC1polynucleotide operably linked to a constitutive promoter as follows. Toinduce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, arecultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising the ZmSNAC sequence asdescribed above can be inserted into a unique restriction site of thevector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M) and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 3 Rice Transformation

One method for transforming DNA into cells of higher plants that isavailable to those skilled in the art is high-velocity ballisticbombardment using metal particles coated with the nucleic acidconstructs of interest (see, Klein, et al., Nature (1987) (London)327:70-73 and see, U.S. Pat. No. 4,945,050). A Biolistic PDS-1000/He(BioRAD Laboratories, Hercules, Calif.) is used for thesecomplementation experiments.

The bacterial hygromycin B phosphotransferase (Hpt II) gene fromStreptomyces hygroscopicus that confers resistance to the antibiotic maybe used as the selectable marker for rice transformation. In the vector,the Hpt II gene may be engineered with the 35S promoter from CauliflowerMosaic Virus and the termination and polyadenylation signals from theoctopine synthase gene of Agrobacterium tumefaciens. For example, seethe description of vector pML18 in WO 97/47731, published on Dec. 18,1997, the disclosure of which is hereby incorporated by reference.

Embryogenic callus cultures derived from the scutellum of germinatingrice seeds serve as source material for transformation experiments. Thismaterial is generated by germinating sterile rice seeds on a callusinitiation media (MS salts, Nitsch and Nitsch vitamins, 1.0 mg/l 2,4-Dand 10 μM AgNO₃) in the dark at 27-28° C. Embryogenic callusproliferating from the scutellum of the embryos is transferred to CMmedia (N6 salts, Nitsch and Nitsch vitamins, 1 mg/l 2,4-D, Chu, et al.,(1985) Sci. Sinica 18:659-668). Callus cultures are maintained on CM byroutine sub-culture at two-week intervals and used for transformationwithin 10 weeks of initiation.

Callus is prepared for transformation by subculturing 0.5-1.0 mm piecesapproximately 1 mm apart, arranged in a circular area of about 4 cm indiameter, in the center of a circle of Whatman® #541 paper placed on CMmedia. The plates with callus are incubated in the dark at 27-28° C. for3-5 days. Prior to bombardment, the filters with callus are transferredto CM supplemented with 0.25 M mannitol and 0.25 M sorbitol for 3 hr inthe dark. The petri dish lids are then left ajar for 20-45 minutes in asterile hood to allow moisture on tissue to dissipate.

Each genomic DNA fragment is co-precipitated with pML18 (containing theselectable marker for rice transformation) onto the surface of goldparticles. To accomplish this, a total of 10 μg of DNA at a 2:1 ratio oftrait:selectable marker DNAs are added to 50 μl aliquot of goldparticles that have been resuspended at a concentration of 60 mg ml⁻¹.Calcium chloride (50 μl of a 2.5 M solution) and spermidine (20 μl of a0.1 M solution) are then added to the gold-DNA suspension as the tube isvortexing for 3 min. The gold particles are centrifuged in a microfugefor 1 sec and the supernatant removed. The gold particles are washedtwice with 1 ml of absolute ethanol and then resuspended in 50 μl ofabsolute ethanol and sonicated (bath sonicator) for one second todisperse the gold particles. The gold suspension is incubated at −70° C.for five minutes and sonicated (bath sonicator) if needed to dispersethe particles. Six μl of the DNA-coated gold particles are then loadedonto mylar macrocarrier disks and the ethanol is allowed to evaporate.

At the end of the drying period, a petri dish containing the tissue isplaced in the chamber of the PDS-1000/He. The air in the chamber is thenevacuated to a vacuum of 28-29 inches Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1080-1100 psi. Thetissue is placed approximately 8 cm from the stopping screen and thecallus is bombarded two times. Two to four plates of tissue arebombarded in this way with the DNA-coated gold particles. Followingbombardment, the callus tissue is transferred to CM media withoutsupplemental sorbitol or mannitol.

Within 3-5 days after bombardment the callus tissue is transferred to SMmedia (CM medium containing 50 mg/l hygromycin). To accomplish this,callus tissue is transferred from plates to sterile 50 ml conical tubesand weighed. Molten top-agar at 40° C. is added using 2.5 ml of topagar/100 mg of callus. Callus clumps are broken into fragments of lessthan 2 mm diameter by repeated dispensing through a 10 ml pipet. Threeml aliquots of the callus suspension are plated onto fresh SM media andthe plates are incubated in the dark for 4 weeks at 27-28° C. After 4weeks, transgenic callus events are identified, transferred to fresh SMplates and grown for an additional 2 weeks in the dark at 27-28° C.

Growing callus is transferred to RM1 media (MS salts, Nitsch and Nitschvitamins, 2% sucrose, 3% sorbitol, 0.4% Gelrite® gelling agent+50 ppmhyg B) for 2 weeks in the dark at 25° C. After 2 weeks the callus istransferred to RM2 media (MS salts, Nitsch and Nitsch vitamins, 3%sucrose, 0.4% Gelrite® gelling agent+50 ppm hyg B) and placed under coolwhite light (˜40 μEm⁻²s⁻¹) with a 12 hr photoperiod at 25° C. and 30-40%humidity. After 2-4 weeks in the light, callus begin to organize, andform shoots. Shoots are removed from surrounding callus/media and gentlytransferred to RM3 media (½×MS salts, Nitsch and Nitsch vitamins, 1%sucrose+50 ppm hygromycin B) in Phytatrays™ culture vessels (SigmaChemical Co., St. Louis, Mo.) and incubation is continued using the sameconditions as described in the previous step.

Plants are transferred from RM3 to 4″ pots containing Scotts MetroMix®350 growing medium after 2-3 weeks, when sufficient root and shootgrowth have occurred.

Example 4 Variants of ZmSNAC

A. Variant ZmSNAC Nucleotide Sequences that do not Alter the EncodedAmino Acid Sequence

The ZmSNAC nucleotide sequences set forth in SEQ ID NO: 1, 3, 5, 7 and 9are used to generate variant nucleotide sequences having the nucleotidesequence of the open reading frame with about 70%, 75%, 80%, 85%, 90% or95% nucleotide sequence identity when compared to the correspondingstarting unaltered ORF nucleotide sequence. These functional variantsare generated using a standard codon table. While the nucleotidesequence of the variant is altered, the amino acid sequence encoded bythe open reading frame does not change.

B. Variant Amino Acid Sequences of ZmSNAC1-5

Variant amino acid sequences of ZmSNAC1, ZmSNAC2, ZmSNAC3, ZmSNAC4 andZmSNAC5 are generated. In this example, one or more amino acids arealtered. Specifically, the open reading frame set forth in SEQ ID NO: 2,4, 6, 8 or 10 is reviewed to determine the appropriate amino acidalteration. The selection of an amino acid to change is made byconsulting a protein alignment with orthologs and other gene familymembers from various species. See, FIG. 1. An amino acid is selectedthat is deemed not to be under high selection pressure (not highlyconserved) and which is rather easily substituted by an amino acid withsimilar chemical characteristics (i.e., similar functional side-chain).Assays as outlined elsewhere herein may be followed to confirmfunctionality. Variants having about 70%, 75%, 80%, 85%, 90% or 95%nucleic acid sequence identity to each of SEQ ID NO: 2, 4, 6, 8 and 10are generated using this method.

C. Additional Variant Amino Acid Sequences of ZmSNAC Polypeptides

In this example, artificial protein sequences are created having 80%,85%, 90% and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom the alignment set forth in FIG. 1 and then the judiciousapplication of an amino acid substitutions table. These parts will bediscussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among the ZmSNAC proteins or amongthe other ZmSNAC polypeptides. See, FIG. 1. Based on the sequencealignment, the various regions of the ZmSNAC polypeptides that canlikely be altered can be determined. It is recognized that conservativesubstitutions can be made in the conserved regions without alteringfunction. In addition, one of skill will understand that functionalvariants of the ZmSNAC sequence of the invention can have minornon-conserved amino acid alterations in the conserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 1.

First, any conserved amino acids in the protein that should not bechanged are identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C and P are not changed. The changes will occur with isoleucinefirst, sweeping N-terminal to C-terminal. Then leucine and so on downthe list until the desired target is reached. Interim numbersubstitutions can be made so as not to cause reversal of changes. Thelist is ordered 1-17, so start with as many isoleucine changes as neededbefore leucine, and so on down to methionine. Clearly many amino acidswill in this manner not need to be changed. L, I and V will involve a50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof ZmSNAC 1-5 are generated having about 82%, 87%, 92% and 97% aminoacid identity to the starting unaltered ORF nucleotide sequence of SEQID NO: 2, 4, 6, 8 or 10.

TABLE 1 Substitution Table Strongly Rank of Similar and Order AminoOptimal to Acid Substitution Change Comment I L, V 1 50:50 substitutionL I, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L17 First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

Example 5 Amplification of Additional ZmNAC Genes from Soybean or OtherPlant Species

Additional ZmNAC or ZmSNAC genes from plant species could be identifiedby PCR or RT-PCR methods using degenerate primers such as the onesdescribed below. Degenerate primers can be designed against conservedamino acid motifs found in available ZmNAC proteins from soybean, maize,rice or Arabidopsis. Such motifs can be identified from an alignment ofthe protein sequences. Sense/antisense primers could be used indifferent combinations. Similarly, several rounds of PCR could be used.The product of amplification of one pair of sense/antisense primerscould be used as template for PCR with another set of internal (nested)degenerate primers therefore maximizing the chances for amplification ofan appropriate sequence, i.e., containing a sequence corresponding tothe corresponding amino acid motif.

Example 6 Transformation of Zea mays by Agrobacterium

For Agrobacterium-mediated transformation of maize, the method of Zhaois employed (U.S. Pat. No. 5,981,840, and PCT Patent PublicationWO98/32326, the contents of which are hereby incorporated by reference).Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the expression cassette to at least one cell ofat least one of the immature embryos (step 1: the infection step). Inthis step the immature embryos are immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos areco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos are cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos are incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos are cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.

Example 7 Activity of the ZmSNAC1 Promoter

To demonstrate that the DNA sequence isolated as the ZmSNAC1 promoterfunctions as a promoter, transgenic maize assays were performed. Theseassays provided a rapid assessment of whether the DNA sequence tested isable to direct gene expression.

The full length promoter (see, SEQ ID NO: 11) was PCR amplified fromgenomic DNA and cloned in an expression cassette as a translationalfusion with B-glucuronidase (GUS; see, Jefferson, et al., (1987) EMBO J16:3901 and U.S. Pat. No. 5,599,670). Transgenic plants were created byAgrobacterium-mediated transformation (see, Example 7) of the expressioncassette into rapid-cycling maize as described in US Patent ApplicationPublication 2003/0221212.

Leaf or stalk tissue was excised from T0 plants for analysis of ZmSNAC1promoter activity. GUS expression was determined by immersing theexcised plant tissues in either (1) a GUS staining buffer modified fromJefferson, et al., (1987 Plant Mol. Biol. Rep. 5:387-405) containing1.36 g NaH₂PO₄, 1.74 g Na₂HPO₄, 164 mg K₄Fe(CN)₆3H₂O, 211 mg K₃Fe(CN)₆,0.06 ml Triton X-100 and 50 mg X-Gluc (Sodium Salt) in a final volume of100 ml or (2) a GUS staining buffer of McCabe, et al., (McCabe andMartinell, (1993) Bio/Technol. 11:596-598) containing 1.36 g NaH₂PO₄,1.74 g Na₂HPO₄, 16.4 mg K₄Fe(CN)₆3H₂O, 0.29 g EDTA, 0.2 ml Triton X-100and 50 mg X-Gluc (Sodium Salt) in a final volume of 100 ml. The planttissue was incubated in the dark overnight at 37° C. Replacing the GUSstaining solution with 70% ethanol stopped the assay. GUS activity wasquantified using plant extract and expressed as nmoles/mg totalprotein/hour; see, Côté and Rutledge, (2003) Plant Cell Rep21(6):619-624. Ten non-transgenic control events were also tested. Sevenof the ten transgene-positive events (Events 1.1, 1.2, 1.3, 1.4, 2.5,1.7, 1.10) showed significant GUS staining in a range of activitylevels; see, FIG. 2. The ten control events did not show any significantGUS staining.

To further confirm that the ZmSNAC1 promoter can be induced by stress,excised tissue was incubated in 5 μM abscisic acid (ABA) for 16 hoursand GUS expression was again evaluated as described above. ABA treatmentresulted in increased staining in the seven previously-identifiedevents, as shown in FIG. 2.

These data confirm the promoter function and stress-inducible nature ofSEQ ID NO: 11 and its endogenous gene ZmSNAC1.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. An isolated polypeptide having transcription factor activity andcomprising an amino acid sequence at least 95% identical to SEQ ID NO:2, 4, 6, 8 or
 10. 2. An isolated polynucleotide comprising a nucleotidesequence at least 95% identical to SEQ ID NO: 1, 3, 5, 7 or 9, or thecomplement thereof.
 3. A transgenic plant comprising a polynucleotide ofclaim 2 operably linked to a promoter that drives expression in theplant, wherein transcription factor activity in said plant is modulatedrelative to a control plant.
 4. The plant of claim 3, wherein saidtranscription factor activity is increased.
 5. The plant of claim 3,wherein said promoter is a tissue-preferred promoter, a constitutivepromoter, or an inducible promoter.
 6. The plant of claim 3 wherein saidtissue-preferred promoter is a root-preferred promoter, a leaf-preferredpromoter, a shoot-preferred promoter or an inflorescence-preferredpromoter.
 7. The plant of claim 3, wherein said modulation oftranscription factor activity affects floral development.
 8. The plantof claim 3, wherein said modulation of transcription factor activityaffects root development.
 9. The plant of claim 3, wherein the stresstolerance of said plant is increased relative to a control.
 10. Theplant of claim 9, wherein the drought tolerance of said plant isincreased relative to a control.
 11. The plant of claim 3, wherein saidpromoter is stress-induced.
 12. A transformed seed of the plant of claim3.
 13. The plant of claim 3, wherein said plant is maize, wheat, rice,barley, sorghum, rye, soybean, brassica or sunflower.
 14. A plant inwhich expression of a native genomic locus is modified, said genomiclocus comprising a polynucleotide of claim
 2. 15. A method of modulatingtranscription factor activity in a plant, comprising transforming saidplant with a polynucleotide of claim 2 operably linked to a promoter.16. The method of claim 15 wherein said modulation of transcriptionfactor activity improves stress tolerance.
 17. The method of claim 15wherein said promoter is a tissue-preferred or inducible promoter, or isboth tissue-preferred and inducible.
 18. The method of claim 17, whereinsaid promoter is stress-inducible.
 19. The method of claim 17, whereinsenescence is delayed.
 20. An isolated nucleic acid molecule comprisinga first polynucleotide which initiates transcription of a second,operably-linked polynucleotide in a plant cell, wherein the firstpolynucleotide is selected from the group consisting of: a) SEQ ID NO:11; b) at least 500 contiguous nucleotides of SEQ ID NO:
 11. 21. Anexpression cassette comprising the first and second polynucleotides ofclaim
 20. 22. A plant cell having stably incorporated into its genomethe expression cassette of claim
 21. 23. A plant having stablyincorporated into its genome the expression cassette of claim 21.